Molecular Regulation Mechanism Of PuMYB40 On Low Phosphorus-induced Adventitious Root Formation In Populus Ussuriensis

Root system is the most important organ to absorb phosphorus,when plants suffer from phosphate starvation,plant root system can change its architecture to cope with low phosphate stress.However,limited information is available about the low phosphate signaling regulates adventitious root formation and transcriptional regulation mechanisms in poplars in response to Pi deficiency.Therefore,it is very urgent and necessary to clarify the mechanism of root system response to low phosphate condition in poplar.(1)New cuttings from in vivo Populus ussuriensis were pre-cultured on normal Pi(200μM KH2PO4)WPM medium for five days(adventitious root primordium emerged),then transferred to low Pi(10 μM KH2PO4)WPM medium.Phenotype analysis showed the number of adventitious roots under low phosphate condition was bigger than that under normal phosphate condition in Populus ussuriensis,indicated that phosphate starvation promoted the adventitious root formation at early stage.(2)Tissue-cultured segments were pre-cultured under normal phosphate condition for five days,then treated with low phosphate stress for 12 h.Collected 0.5 cm of the stem basal,which contained adventitious root primordium to perform paraffin sections.The results showed that low phosphate condition induced more new adventitious root primorediums formation to produce more adventitious roots than that under normal phosphate condition.(3)Based on the results of root phenotype analysis under low phosphate condition,pre-cultured P.ussuriensis for five days under normal phosphate condition to treated with low phosphate(0h).Then we collected the adventitious root primorediums using 11#knife blade to perform time-course RNA-seq at five time points(2h,4h,8h,12h and 24h).The adventitious root primorediums from normal phosphate condition at corresponding time point served as control.Totally,we obtained 1706 DEGs,including 817 up-regulation and 889 down-regulation.GO analysis showed that for "Molecular Function","DNA-binding transcription factor activity(GO:0003700)" enriched the most DEGs.Otherwise,"response to stimulus"(GO:0050896)enriched the most DEGs.(4)Then we selected 29 low phosphate-,auxin-and root development-related deferentially expressed genes as bottom genes to construct a multi-layered hierarchical regulatory network(ML-hGRN)based on GO annotation using Bottom-up GGM method.PuMYB40 got the highest interference frequency in Layer 3 and may play an important role in low phosphate-induced adventitious root formation.(5)Homology analysis showed PuMYB40 belongs to typical R2R3-type MYB transcription factor.Transcription activation assay showed that PuMYB40 possessed the transcriptional activation activity and the site of transcriptional activation activity was in C terminal region(109-282 aa).(6)PuMYB40 was significantly up-regulated during adventitious root formation under early low phosphate condition.Throughout Agrobacterium-mediate leaf disc method,we obtained 19 PuMYB40-OE lines and 16 PuMYB40-SRDX lines.Comparing to WT under normal phosphate condition,PuMYB40-OE lines showed early about 1 day root emergence and possessed more adventitious roots in number.PuMYB40-SRDX showed opposite phenotype.The number of adventitious roots and root biomass showed no obviously change low phosphate and normal phosphate condition in PuMYB40-SRDX.These results suggesting that PuMYB40 may take an important part in early low phosphate-induced adventitious root formation.(7)According to our constructed ML-hGRN,PuLRPl and PuERF003 acted as the target genes of PuMYB40 and PuWRKY75.The results of RT-qPCR showed that the relative expression level of PuLRP1 and PuERF003 was significantly up-regulated comparing to WT in PuMYB40-OE and PuWRKY75-OE lines.While,the relative expression level of PuLRP1 and PuERF003 was significantly down-regulated comparing to WT in PuMYB40-SRDX and PuWRKY75-SRDX lines.Then Yeast one-hybrid,ChIP-qPCR and EMSA assays showed that PuLRPl and PuERF003 were the directly target genes of PuMYB40 and PuWRKY75.LUC assay confirmed that PuMYB40 interact with PuWRKY75 to promote PuLRP1 and PuERF003 transcription.(8)The adventitious root number of PuLRP1-OE and PuERF003-OE lines was larger than that in WT.The adventitious root number of PuLRP1-SRDX and PuERF003-SRDX lines was smaller than that in WT.The adventitious roots number and root biomass of PuLRP1-SRDX and PuERF003-SRDX lines showed no significantly change under normal and low phosphate condition.(9)PuWRKY75 belonged to WRKY Ⅱ c subfamily and possesses the secondly highest interference frequency in Layer 3,followed by PuMYB40.Yeast two-hybrid,BiFC and CO-IP assays showed PuMYB40 interacts with PuWRKY75 in vivo and vitro.Moreover,the interact sites were the C-terminal region(109-252 aa)of PuMYB40 and N-terminal region(1-100 aa)of PuWRKY75.Similar to PuMYB40 transgenic lines,the number of adventitious roots in PuWRKY75-OE lines was significantly larger than that in WT.While,PuWRKY75-SRDX lines displayed opposite phenotype.Moreover,the adventitious roots number and root biomass of PuWRKY75-SRDX lines showed no significantly change under normal and low phosphate condition.This study can provide important theoretical basis and genetic resources for the analysis of molecular regulatory mechanism of adventitious root formation and phosphorus utilization efficiency in poplar.