Elucidation Of The Responsive Mechanisms Of Maize Roots To Long Distance Nitrogen Signals Using A Split Root System

Nitrogen(N)is one of the main elements restricting plant growth and directly affects crop yield and quality.In production,the distribution of nitrogen fertilizer in the soil and the root distribution of crops are usually spatially different due to fertilization methods,root absorption or rain leaching.In the environment,the morphological and structural plasticity of roots growing in regions with sufficient nitrogen is the main mechanism for improving the efficiency of N acquisition and utilization by plants.It is jointly regulated by local signals of the root system and the above-ground long distance signal.Study deeply of the morphological and structural plasticity of maize roots in response to low nitrogen,identification and differentiation of genes in roots that respond to local N signals and long-distance signals,and analysis the signaling pathways,biological processes will help to resolve the molecular mechanism about N uptake and signal regulation of maize.Then it can provide a new idea for improving the utilization efficiency of nitrogen fertilizer in maize research.First,we used high nitrogen(4 mM)as a control to study the morphological and gene-expression differences of three different types of roots at maize seedling stage under N stress(0.5 mM / 0 mM).Then,we used the root separation experiment technique,divided the maize root system into two parts and applied four different concentrations of nitrate to identify the root morphology changes.And the RNA-seq technology was used to comparative analysis the transcriptome of the4 treated roots.In the end,we identified the target genes that regulated by N-long-distance signals in the root system.The main findings are as follows:(1)Different root systems of maize seedlings have different morphological responses to low nitrogen stress.In general,the root length decreases and the root system becomes thinner;the dry weight above the ground decreases and the root-to-root ratio increases;the axial root grows and the lateral root becomes shorter.The response of different types of root systems to low nitrogen is significantly different,total root length and lateral root length of primary root(PR)or crown root(CR),the surface area,volume,and dry weight are reduced,and the PR performance is more significant,the growth and development of seminal root(SR)are enhanced;the spatial distribution of the lateral roots of the three types of root systems is very obvious.The number of lateral roots in the same section under low nitrogen increased in SR and CR,and remained basically unchanged in PR.(2)Three types of root systems have different gene expression patterns.RNA-seq analysis revealed that the gene expression patterns between the PR and SR were similar,but the CR between PR was quite different,that highlighting the specific development of CR.Under high nitrogen and nitrogen deficiency conditions,1445 and 1978 differentially expressed genes were detected between different root systems.Their functions mainly involved anatomical structure development,secondary metabolic processes,transcription factor activity,and oxidoreductase activity.(3)Different root systems of maize seedling stage respond differently to low nitrogen stress.We identified 2555,1896,and 1781 differentially expressed genes in PR,SR,and CR in response to nitrogen deficiency stress,of which 1126 were shared DEGs.Various N-response-related genes are expressed dynamically between roots and under two treatments.Some genes such as NRT1.1B,NRT2.5,NRT2.7,NAC4,CKX6,AP2-EREBP181 respond to nitrogen deficiency stress in three types of roots.And the expression trends are consistent,while other genes such as AP2-EREBP205,AP2-EREBP54,NAC122,NAC60,NLP7,and ZNF2 have different trends in response to nitrogen deficiency in different root systems which called root-specific.(4)The maize root system has morphological plasticity that tends toward N growth.Heterogeneous nitrate supply regulates root development.Root growth on the high nitrogen side is significantly compensated by the effect of low nitrogen supply on the other side,while the root system on the low nitrogen side is affected by the high nitrogen supply on the other side.It is more sensitive to the "N demand" signal induced by low nitrogen environment.(5)Different maize roots system respond differently to low N-long-distance signals.Using RNA-seq to comparative analysis the transcriptome of roots under average and local high nitrogen,then we identified 707,1208,and 24133 genes in PR,SR,and CR respectively,which in response to the long-distance signal of "N demand",and 344 genes are shared by the three types of root systems,whose functions mainly involve the regulation of biological processes,the regulation of nucleic acid transcription,and ion binding.These genes include b HLH99,ERF-like,CIPK-like,NAC2,LOB42 and other gene family members related to N response.Genes consistent with their expression trends include Zm00001d040682,Zm00001d006170,Zm00001d009160,Zm00001d002161,etc.They are strong candidates for "N demand" long-range signal target genes.(6)Different maize root systems respond differently to high N long-distance signals.Comparing the transcriptomes of root systems with both nitrogen deficiency and local nitrogen deficiency,1169,1259,and 838 genes were identified in PR,SR,and CR,respectively,that in response to the long-distance signal of "N supply",of which 357 were shared by three types of roots.Theirs function mainly involves flavonoid metabolism process,redox process,nucleic acid binding transcription factor activity,etc.These genes include NRT2.7,GRX,MYB,bZIP,AP2-EREBP and other gene family members related to N response.Genes consistent with their expression trends include Zm00001d038352,Zm00001d039648,Zm00001d052673,Zm00001d012394,etc.They are candidates for "N supply" long-distance signaling target genes.In summary,different maize root systems have different gene expression patterns and different responses to low nitrogen and N long-distance signals.This provides a new perspective on the study of a single root type for a deep understanding of the root response to the environment.The identification of target genes about long-distance signals has promoted the understanding of the molecular mechanism of signal regulation of maize N response,thus provided new ideas for improving the nitrogen utilization efficiency of maize.