Transcriptional And Translational Regulatory Mechanisms Of Rice Nitrate Transporter Gene OsNRT2.3a/b

The negative impact of climate change on crop yield is being witnessed,agricultural sustainability must be ensured under global warming.Projected global warming poses major risks to agricultural systems by reducing the global yields of major crops.There has been reported that increased nighttime temperature severely affected nitrogen uptake and use efficiencies and significantly reduced rice growth and yield.Therefore,we must ensure the sustainability of agriculture in the face of global warming.After a long period of natural evolution and human selection,rice has evolved two different absorption nitrate mechanisms.The molecular biological functions of OsNRT2.3a and OsNRT2.3b which belonging to high-affinity nitrate transporters have gradually been discovered.The isoforms OsNRT2.3a and OsNRT2.3b are two transcripts with H⁺-coupled nitrate（NO₃^-）transport produced by the gene OsNRT2.3.OsNRT2.3a is mainly responsible for nitrate in the xylem from root to stem.OsNRT2.3b,contains an intron,is mainly expressed in the phloem and contributes to the p H and ion homeostasis of the phloem.Knockdown of the OsNRT2.3a in rice can increase the accumulation of NO₃^-in the roots,but the overexpressed OsNRT2.3b can increase the nitrogen uptake of plants and increase the yield of rice.However,the regulatory mechanism of OsNRT2.3 gene being splicing into isoform OsNRT2.3a and OsNRT2.3b and its relationship with temperature regulation are still unclear.In this study,we explored the key nucleotide variations that can affect the alternative splicing of OsNRT2.3 into two isoforms of OsNRT2.3a and OsNRT2.3b and the regulatory effects of high and low temperature on OsNRT2.3a and OsNRT2.3b by using natural mutant lines and TILLING backcross lines.Furthermore,we also identified a TATA-box binding protein（OsTBP2.1）which exhibited strong binding to the TATA-box of OsNRT2.3 through yeast one-hybrid.Also,two sRNAs（s NRT2.3-1 and s NRT2.3-2）can regulated the transcription and translation of OsNRT2.3a and OsNRT2.3b in a temperature-dependent manner were obtained by sRNA sequencing.The specific research results are as follows:1.The rice varieties with nitrogen selection pressure were treated with high and low nitrogen,and the expression level of OsNRT2.3a and OsNRT2.3b was analyzed by q RT-PCR.The results showed that there was a certain linear relationship between the transcription ration of isforms OsNRT2.3b/a and nitrogen content in rice under low nitrogen treatment,rather than high nitrogen treatment.2.According to the average minimum temperature and maximum temperature in the four years from 2017 to 2020,the plant height,yield and biomass of Hap A,Hap B and Hap C were statistically analyzed under field conditions.The results showed that the plant height,yield and biomass of Hap B were significantly increased compared with Hap A and Hap C,and the yield and biomass of Hap B were still maintained even under high temperature conditions in 2018.Also,the Hap A and Hap B plants were treated with high and low temperatures under hydroponic conditions.The transcription and translation efficiency of the isoforms of OsNRT2.3a and OsNRT2.3b were then analyzed.Compared with Hap A,the translation efficiency of OsNRT2.3a and OsNRT2.3b in natural variants of Hap B was increased significantly,but high temperature decreased the OsNRT2.3a protein lifetime,while its effect on OsNRT2.3b protein was not obvious.3.In order to verify the effect of mutation on the cis-acting element TATA-box（from TATA to TACA）residing at 83 bp upstream from the ATG start codon on the transcriptional regulation of OsNRT2.3 gene,the transgenic lines of p NRT2.3:OsNRT2.3a and mp NRT2.3:OsNRT2.3a,p NRT2.3:OsNRT2.3b and mp NRT2.3:OsNRT2.3b were obtained.q RT-PCR analysis showed that the expression of OsNRT2.3b was increased in the lines of mp NRT2.3:OsNRT2.3a and mp NRT2.3:OsNRT2.3b,but the expression of OsNRT2.3a was repressed in the lines of mp NRT2.3:OsNRT2.3a.It is suggested that the cis-acting element TATA-box of OsNRT2.3 plays a key regulatory role in the OsNRT2.3 transcription process.4.The four-year average minimum temperature from 2017 to 2020 was conjoint analyzed with the yield and ANUE of wild-type（WT）and TILLING backcross lines（T11/WT（aa）and T12/WT（aa））in field.Also,the nitrogen uptake of WT,T11/WT（aa）and T12/WT（aa）at high and low temperatures were carried out under hydroponic conditions.The results showed that T11/WT（aa）and T12/WT（aa）have stronger tolerance to high temperature given that these lines exhibited increasing yield,nitrogen absorption and ANUE compared with WT.Western blotting showed that the protein level of OsNRT2.3a and OsNRT2.3b was increased in T11/WT（aa）and T12/WT（aa）.Furthermore,it is observed that high temperature can decrease the lifetime of OsNRT2.3a protein,but it has no significant effect on OsNRT2.3b protein,which is similar to the observation in Hap B.5.The results of yeast one-hybrid showed that the protein OsTBP2.1 can bind to TATA-box residing at-83 bp upstream from the ATG start codon of OsNRT2.3 gene.By constructing a fluorescent expression vector of Fluc/Rluc in transient expression experiments,it is shown that the TATA-box binding protein OsTBP2.1 can promote the translation efficiency of Fluc protein by binding to the-83 bp upstream of the ATG start codon of OsNRT2.3 gene,but the function is lost when TATA-box was mutated to TACA-box.6.The molecular function of s NRT2.3-1/-2 was verified by Northern blot,in vitro experimental verification and transient expression experiment.Field experiments of Hap B and TILLING backcross lines were also conducted to verify the biological function of s NRT2.3-1/-2.The results showed that the OsNRT2.3a intron and low temperature treatment can promote the binding between s NRT2.3-1/-2 and OsNRT2.3a,and that s NRT2.3-1 may be formed from the degradation of OsNRT2.3a/b.The expression of s NRT2.3-1/-2 is higher in high temperature condition than in low temperature condition,which may be the result of the higher degradation rate of OsNRT2.3a under high temperature condition and the temperature effect on the complex RNA secondary structure of OsNRT2.3a.In addition,the expression of s NRT2.3-1/-2 was reduced in Hap B and TILLING backcross lines,which led to increased translation efficiency of OsNRT2.3a and OsNRT2.3b.In summary,a novel haplotype（Hap B）of at rice nitrate transporter OsNRT2.3 gene and TILLING lines have been discovered,which can maintain high yield and high nitrogen use efficiency under high temperature condition.We also found that rice tolerance to high temperature is positively correlated with the translation of OsNRT2.3b m RNA isoform and negatively correlated with the abundance of a unique sRNA（s NRT2.3-1）,which derived from5’untranslated region of OsNRT2.3 and binded to OsNRT2.3a in a temperature-dependent manner.In addition,OsTBP2.1 binding protein could regulate the transcription of genes OsNRT2.3 to OsNRT2.3a and OsNRT2.3b.Our findings revealed that allelic variations of the5’untranslated region of OsNRT2.3 increase the OsNRT2.3b protein lifetime during high-temperature stress.The finding provided a breeding strategy for improving grain yield and enhancing adaptation of crops to greenhouse warming in the future.