Functional Characterization Of Central Rhythm Factor Nhd1 In Regulating Root Growth And Utilization Of Carbon And Nitrogen In Rice

Both nitrogen and carbon are important for plant growth and development.Ammonium（NH₄⁺）and nitrate（NO₃^-）are the main inorganic nitrogen sources absorbed by plant from soil,and they entry into the root is mediated by ammonium and nitrate transporters.Carbohydrate,especially sucrose,as the main product of photosynthesis,are the main form of long-distance transport in plants,providing energy and carbon skeletons in various biosynthetic processes.The long-distance transport of sucrose in the phloem must be assisted by sucrose transporter proteins localized in the cell membrane.The metabolic processes of carbon and nitrogen are closely related and highly dependent on each other.Plant root formation is regulated by several factors,and it is generally accepted that nitrate regulates root elongation,while ammonium regulates root branching.Plants are able to adapt to different environments by regulating root structure and the activity of nitrogen transporters.The importance of carbon and nitrogen balance has been confirmed by multiple studies,and the process of carbon and nitrogen metabolism is regulated by rhythms.In rice,we have previously reported that a biological clock rhythm factor N-mediated heading date-1（Nhd1）can regulate flowering time.In this study,molecular screening of the Nhd1-regulated ammonium transporter gene AMT1.3 involved in root response to different concentrations and forms of nitrogen and NRT2.4 involved in nitrate distribution,identification of the Nhd1-regulated sucrose transporter gene SUT1.The molecular mechanism and physiological function of Nhd1 regulating AMT1.3,NRT2.4 and SUT1 were studied via genetic,physiological and molecular biological analysis.The main results obtained were as follows:1.Determination of root growth and ¹⁵N uptake rate revealed that mutations in the transcription factor Nhd1 resulted in impaired root growth with nitrate or low ammonium（0.25 m M NH₄⁺）supply,and reduced ¹⁵N-NH₄⁺uptake rate with low ammonium supply.Analysis of nitrogen transporter gene promoter elements and RT-q PCR revealed that AMT1.3 and NRT2.4 promoters contain Nhd1 binding-sites and their expression is substantially suppressed in leaves and roots due to Nhd1 mutation.The results of yeast-one hybrid assay,electrophoretic mobility shift assay（EMSA）,chromatin immunoprecipitation（Ch IP）-q PCR assay and transient transactivation assay indicated that Nhd1 could directly bind to AMT1.3 and NRT2.4 promoters.2.The results of immunostaining showed that in seminal root,Nhd1 was expressed in the exodermis,cortex,pericycle,vascular parenchyma;in lateral root,Nhd1 was expressed in exodermis,sclerenchyma,pericycle and vascular parenchyma cells.AMT1.3 was specifically expressed in the phloem companion cells in seminal root and expressed in the sclerenchyma cell layer,pericycle and vascular parenchyma cells in lateral root.Nhd1co-localized with AMT1.3 in all the cell types showing AMT1.3 expression.3.Characterization of the physiological function of AMT1.3 using the Xenopus laevis oocyte transport system showed that the uptake of ammonium by AMT1.3 c RNA-injected Xenopus laevis oocytes increased 1-fold compared with the water-injected control,whereas the uptake of ammonium by AMT1.3 c RNA-injected Xenopus laevis oocytes did not differ from the control when they were incubated in 2.5 m M ¹⁵N-NH₄⁺solution.In addition,the Km value of ¹⁵N-NH₄⁺uptake by AMT1.3 c RNA-injected Xenopus laevis oocytes under low concentration conditions was 63μM.These results indicate that AMT1.3 is a high-affinity ammonium transporter protein.It was found by ¹⁵N uptake rate assay that nhd1 and amt1.3mutants showed similar reduced transient uptake rate phenotypes at 0.25 m M ¹⁵N-NH₄⁺supply,while nhd1 and amt1.3 mutants phenotypes did not differ from wild type at¹⁵N-NO₃^-or 2.5 m M ¹⁵N-NH₄⁺supply.Overexpression of AMT1.3 in nhd1 restored its uptake of low ammonium concentration to wild-type levels.4.¹⁵N distribution results showed that mutation of either Nhd1 or NRT2.4 at 5 m M¹⁵N-NO₃^-supply resulted in increased nitrogen content in basal nodes and leaf sheaths and decreased content in leaves.The results of the ¹⁵N-NO₃^-redistribution among different leaves showed that when 5 m M ¹⁵N-NO₃^-was supplied to the leaves,mutations in Nhd1 and NRT2.4 resulted in an increase of nitrogen in the basal node and a decrease in the 1^stleaf,and overexpression of NRT2.4 in nhd1 mutant restores this defect.5.Knockdown of Nhd1 or NRT2.4 at nitrate supply resulted in a phenotype of shorter seminal root and reduced lateral root length and number,whereas knockdown of Nhd1 or AMT1.3 at low ammonium supply resulted in a phenotype of shorter seminal root and reduced lateral root length and number.Knockout of Nhd1,AMT1.3 and NRT2.4 all resulted in a shorter root and reduced root dry weight phenotype under low nitrogen conditions（150kg-N/ha）in the field,while root growth did not differ from the wild type in high nitrogen fields.This is due to the fact that ammonium is the main nitrogen source in high nitrogen（300 kg-N/ha）paddy,and the consistently high proportion of ammonium may have weakened the response of roots of the nrt2.4 mutants to nitrate,whereas in low nitrogen paddy,the strong nitrification and rapid uptake of ammonium by roots due to the low nitrogen content resulted in rice roots that may experience a relatively high proportion of NO₃^-.6.Compared with wild type and negative control,nhd1 and nrt2.4 mutants showed significantly lower plant height and total nitrogen accumulation but more tillers at 56-91days after seedling emergence,regardless of nitrogen supply.Meanwhile,knockout of AMT1.3 suppressed plant height and reduced nitrogen accumulation only under low nitrogen supply.The knockout of Nhd1 resulted in a two-week delay in flowering,therefore,the nutritional growth period and nitrogen uptake period of the nhd1 mutant were prolonged,allowing the nhd1 mutant to accumulate more total nitrogen and obtain higher nitrogen uptake efficiency than the wild type at maturity under low nitrogen paddy.Under low nitrogen conditions,Nhd1 knockout resulted in increased yield,while NRT2.4knockout resulted in decreased grain yield,and grain yield of the amt1.3 mutant was not significantly different from the wild type under either high or low nitrogen conditions.Overexpression of NRT2.4 increased total nitrogen accumulation and nitrogen uptake efficiency of nhd1 mutants at high or low nitrogen levels,whereas overexpression of AMT1.3 increased total nitrogen accumulation and nitrogen uptake efficiency only under low nitrogen conditions.7.Phenotypic assays showed that the mutation of Nhd1 resulted in dwarfing of the plants and reduced dry weight.Sucrose content in leaf sheaths and leaf blades was measured separately at the end of light,and it was found that the mutation of Nhd1 only reduced the sucrose content in leaf sheaths but did not change the sucrose content in leaf blades.In addition,Nhd1 mutation led to an increase in carbon/nitrogen ratio,a decrease in photosynthetic rate,and a decrease in chlorophyll a/b content in rice.8.RNA-seq results showed that Nhd1 knockdown resulted in a large number of altered gene expressions and significant changes in starch and sucrose metabolism,plant circadian rhythm and amino acid metabolic pathways.RT-q PCR analysis indicated that Nhd1mutation led to a significant increase in the expression of ammonium assimilation genes GS1;1,GS2 and Fd-GOGAT,while expression of nitrate reductase gene NR2 was significantly reduced.In-depth analysis of the effect of Nhd1 on carbon/nitrogen metabolites revealed that in comparison to wild type,the nhd1 mutants had a large amount of altered soluble amino acid content and small molecule organic acid content involved in the tricarboxylic acid cycle.9.The AAA/CAATCT site is an important site for Nhd1 binding,and analysis of the promoter element of know sugar transporter genes and RT-q PCR revealed that the promoter of SUT1 gene contains this site and the expression of SUT1 was suppressed in nhd1mutants.Transactivation assay in rice leaf protoplast cells,Ch IP-q PCR and EMSA experiments further confirmed that Nhd1 can bind directly to the SUT1 promoter and positively activate its expression.10.The sut1 mutants and overexpressed SUT1 plants in nhd1（nhd1/SUT1-OE）were obtained by CRISPR-Cas9 technology and hybridization.The sut1 and nhd1 mutants were found to exhibit similar phenotypes of impaired sucrose transport and reduced plant height and dry weight.Overexpression of SUT1 in the nhd1 mutant fully restored its defect in sucrose transport and partially restored the defects in plant height and dry weight reduction.In summary,we show that Nhd1 can directly activate the expression of a high affinity ammonium transporter gene AMT1.3 and a dual affinity nitrate transporter gene NRT2.4.Physiological,molecular and genetic analysis of nhd1,amt1.3 and nrt2.4 mutants revealed that root growth and nitrogen uptake rate were decreased in nhd1 mutants resulting from the suppression of AMT1.3 and NRT2.4,while total nitrogen accumulation of nhd1 mutants were increased at low nitrogen supply due to a prolonged vegetative and entire growth period,and Nhd1 regulates sucrose distribution and carbon and nitrogen metabolism by interacting with sucrose transporter SUT1.This study further expands the regulatory pathways of nitrogen utilization and flowering in rice,reveals that rhythmic factor can synergistically regulate carbon and nitrogen metabolism and balance,and provides a theoretical basis and candidate genes for future breeding of nutrient-efficient and high-yield varieties using molecular breeding techniques.