Molecular Mechanism Of PuHox52 Response To Low Nitrogen Stress In Populus Ussuriensis

Nitrogen(N)nutrition plays an important role in the growth and development of plants,and the nutrient-poor soil environment is one of the important factors affecting forest yield.The root system is the main way for forest trees to absorb N in the soil.Therefore,understanding the root development mechanism of forest trees in the low-nitrogen environment and cultivating new varieties that are resistant in a low-nitrogen environment are of great significance to the development of forestry.Many studies have shown that the HD-ZIP protein family has an important regulatory role in abiotic stress and hormone response.However,the resistance mechanism of the HD-ZIP family genes under low-nitrogen environment is still rarely reported.The purpose of this study is to carry out the molecular mechanism of the PuHox52,an HD-Zip family I subfamily y-branched,in response to low nitrogen environment in the roots of Populus ussuriensis.(1)The quantitative real time polymerase chain reaction(RT-qPCR)showed that PuHox52 was significantly induced under low nitrogen condition in the roots of P.ussuriensis.Histochemical staining of PuHox52pro::GUS P.ussuriensis transgenic lines showed that visible GUS signal was only detected in leaves with no treated.GUS protein were not only induced in leaves but also in roots,and specifically expressed in lateral roots under low nitrogen condition.(2)The wild-type(WT),overexpression of PuHox52(PuHox52-OE)and repression of PuHox52(PuHox52-RNAi)transgenic lines of P.ussuriensis were subjected to low nitrogen condition and no treated as control,respectively.Under low nitrogen condition,PuHox52-OE transgenic lines increased tolerance compared with WT,the roots of PuHox52-OE transgenic lines were more developed,the root biomass was significantly increased by 63-70%,and the main root was elongated(25-31%increase compared with WT),and the lateral root length and density were increased(55-58%and 28-30%respectively).On the contrary,compared with WT,the root biomass of the PuHox52-RNAi lines decreased by 42%-48%,the main root length decreased by 18%-19%,and the lateral root length and lateral root density were both reduced,significantly reduced by 36-38%and 10-12%respectively.The long-term low nitrogen stress for 60 d experiment on the WT and PuHox52 transgenic lines showed that PuHox52-OE transgenic lines had developed more roots,the biomass increased by 25-27%,and the stronger ability of roots to absorb NO3-than WT,while the root of PuHox52-RNAi lines was reduced by 22-23%compared with WT,and the ability to absorbing NO3-were reduced.These results indicate that the overexpression of PuHox52 can resist the low nitrogen environment by promoting the growth of the lateral roots of the roots and increasing the absorption capacity of the roots to NO3-.(3)Transcriptome sequencing analysis was conducted on the leaves and roots of WT and PuHox52-OE transgenic lines under low nitrogen condition for 3 weeks or no treated as control.We found that many differentially expressed genes(DEGs)between PuHox52-OE and WT in roots under low nitrogen condition,up to 3452 DEGs.Among them,1914 genes were up-regulated,and 1538 genes were down-regulated.Annotated and analyzed these 3452 DEGs,we found that the gene ontology(GO)terms that are significantly enriched in many DEGs are related to nitrogen,including nitrogen utilization,nitrate response,transport and synthesis pathways,nitrogen fixation and glutamine synthesis processes,etc.In addition,GO terms related to phosphate response and transport were also found.GO terms such as water transport and hormone metabolic synthesis pathways were also significantly enriched.(4)In order to verify the target genes that PuHox52 directly binds in response to low nitrogen stress,the yeast one-hybrid(Y1H)experiment was used to verify PuHox52 and 11 candidate target genes related to nitrogen metabolism pathways.The result of Y1H assay indicated that PuHox52 could bind to the promoter region of PuNRT1.1.Chromatin immunoprecipitation(ChIP)experiment was performed on PuHox52-OE transgenic lines.The results of ChIP-PCR and ChIP-qPCR showed that PuHox52 was significantly enriched near the TAATTA element in the promoter region of PuNRT1.1 The electrophoretic mobility shift assay(EMSA)experiment results showed that PuHox52 can specifically bind to the TAATTA element in the promoter region of PuNRT1.1.The above results showed PuHox52 directly binds to the TAATTA cis-element in the promoter region of PuNRT1.1,thereby regulating the expression of PuNRT1.1.(5)Biological analysis of PuNRT1.1 gene sequence of P.ussuriensis showed that PuNRT1.1 belongs to a transmembrane protein.The result of transient transformation of protoplasts showed that PuNRT1.1 was localized on the vacuolar membrane at the cellular level.We obtained 14 PuNRT1.1 overexpression(PuNRT1.1-OE)and 10 repression(PuNRT1.1-RNAi)transgenic lines,and analyzed the phenotypes of WT,PuNRT1.1-OE and PuNRT1.1-RNAi lines under low nitrogen condition and no treated as control.The result showed that compared with WT,PuNRT1.1-OE were promoted the growth of roots under low nitrogen condition,the increase in root biomass was 76.7-78.1%,and the main roots were elongated by 12.8-12.9%compared with WT,and the lateral root length and lateral root density increased significantly,with the increasing ranges of 82-100%and 23.9-27.4%respectively,while the root biomass of PuNRT1.1-RNAi lines decreased significantly under low nitrogen stress,the decrease range was 15.5-19.5%,lateral root length and lateral root density were also significantly reduced,and it was more sensitive to low nitrogen environment.(6)RT-qPCR results showed that PuHDA9 was significantly down regulated in PuHox52-OE transgenic lines and WT during the development of adventitious roots.Meanwhile,PuHDA,were also significantly down regulated in roots of PuHox52-OE transgenic lines under low nitrogen condition compared with WT.We constructed repression of PuHDA9(PuHDA9-RNAi)vector and obtained 12 PuHDA9-RNAi transgenic lines.Phenotypic analysis showed that PuHDA9-RNAi lines can promote the development of adventitious roots.Compared with WT,the number of adventitious roots of the PuHDA9-RNAi lines increased significantly by 35-38%.Y1H,ChIP and EMSA experiments proved that PuHox52 can bind to the cis-element TAATTATT in the promoter region of PuHDA9.The ChIP-seq experiment with H3K9ac antibody showed that PuHox52 enhances the H3K9 acetylation modification level in the TSSs and the coding region of three true target gens of PuHox52(PuWRKY51,PuLBD21 and PuIAA7)by repressed the expression of PuHDA9,thereby increasing this the expression levels of three key regulatory factors indirectly enhance PuHox52-mediated regulation of adventitious root development.Phenotypic analysis of WT and PuHDA9-RNAi lines under low nitrogen stress showed that inhibiting the expression of PuHDA9 could improve the tolerance of P.ussuriensis under low nitrogen stress.The PuHDA9-RNAi line can promote the growth of roots under low nitrogen condition,the biomass of roots increases 83-85%compared with WT.The resistance of PuHDA9 under low nitrogen condition may be related to the negative regulation of PuHDA9 by PuHox52,which affects root development-related genes through H3K9ac modification.In summary,PuHox52 can positively regulate low nitrogen resistance in roots of P.ussuriensis by directly regulate the target gene PuNRT1.1 with promoted the lateral roots and improve the ability of the roots of to absorb NO3-.In addition,PuHox52 regulates the level of H3K9ac modification by inhibiting the expression of PuHDA9,increases the expression of root development genes and promoting root growth,thereby improving the tolerance of P.ussuriensis to low nitrogen stress.This study provides a new idea for the resistance mechanism of poplar roots in a nitrogen-deficient environment and provides genetic resources and a theoretical basis for woody plants to grow in the poorly soiled northeast forest area.