Study On Molecular Mechanism Of OsARFs (Auxin Response Factor) Regulating Nitrogen Metabolism In Rice

Nitrogen is one of the key factors affecting rice yield.The application of nitrogen fertilizer is an important mean to increase grain yield.However,excessive application of nitrogen fertilizer has caused negative effects,such as increased production costs for farmers and environmental pollution.Therefore,improving plant nitrogen use efficiency（NEU）and reducing the use of chemical fertilizers are of great significance for ensuring food security and sustainable agricultural.Plant hormones,including gibberellins,cytokinins,auxins,etc.,mediate the plant regulatory network of nitrogen metabolism.Auxin has always been regarded as a medium that can mediate the transmission of nitrogen signals from the above-ground part to the underground part.In this study,CRISPR/Cas9 technology was used for targeted gene knockout,from which the mutants osarf1 and osarf24 were constructed.This research reveals the molecular mechanism of the transcription factor OsARFs regulating rice nitrate metabolism,and provides important molecular basis for the regulation of plant nitrogen utilization by auxin homeostasis.Key research findings are as following:1.The results of ¹⁵N isotope-labeled nitrate uptake rate show that the nitrate uptake rates of osarf1 and osarf24 are significantly lower than that of wild-type ZH11.Compared with ZH11,osarf1 and osarf24 exhibit reduced plant height,fewer lateral roots and fewer root hairs.In addition,transcription levels of Os NRT1.1B,Os NRT2.3a,Os NPF2.4 and Os NIA2 in osarf1,osarf24 plants were significantly decreased.2.Yeast one-hybrid experiments showed that OsARF1 and OsARF24 could recognize the promoters of genes involved in nitrogen metabolism,such as Os NRT1.1B,Os NRT2.3a,Os NPF2.4 and Os NIA2.EMSA furter showed that OsARF1 and OsARF24 proteins could directly bind to the promoter regions of Os NRT1.1B,Os NRT2.3a,Os NPF2.4 and Os NIA2.3.Transcriptional activation experiments confirmed that OsARF1 and OsARF24proteins have transcriptional activation functions towards downstream genes Os NRT1.1B,Os NRT2.3a,Os NPF2.4 and Os NIA2,and upregulate the expression levels of these genes.4.In order to find potential interacting proteins that work with OsARF1 and OsARF24,OsARF1 and OsARF24 were used as baits to screen the library of yeast two-hybrid（Y2H）.As a result,AIP1 and AIP2 were obtained.The yeast two-hybrid experiment and LUC system were used to further verify the interaction between OsARF1,OsARF24 and AIP1,AIP2,respectively.5.Taking OsARF24 as an example,the transcriptional activation experiments were carried out.The results showed that the interaction of OsARF24 with AIP1 and AIP2 would enhance the activation ability of OsARF24 towards target genes.In summary,the transcription factors OsARF1 and OsARF24 could bind to the promoter regions of downstream target genes Os NRT1.1B,Os NRT2.3a,Os NPF2.4 and Os NIA2.OsARF1 and OsARF24 proteins activate the expression of these genes.AIP1 and AIP2 may play an important role in OsARFs regulating NUE.This study explains the molecular mechanism of how auxin response factors OsARF1 and OsARF24 regulating nitrogen metabolism,which further improves the rice nitrogen metabolic regulation network and provides reference for improving rice NUE.