Rice Nitrate Transport Accessory Protein Gene OsNAR2.1 Participates Lateral Root Formation In A Nitrate-dependent Manner

Rice plants have aerenchymatous tissue in their roots that allow partial oxygen to release into the soil.Thus nitrification occurs immediately in the aerobic niche of the rhizosphere or on the surface of the roots.Therefore,even in a flooded paddy soil,rice roots are actually exposed to the mixture of NH4+ and low NO3-concentration.However,mechanism for NO3-regulating rice root growth is poorly understood,mainly due to ignoring NO3-nutrition in rice plant.Lateral root development in response to NO3-has been investigated extensively in the dicot model plant Arabidopsis.However,our understanding of NO3-regulated LR development is limited in monocot model plant rice.We previously reported that OsNAR2.1,a rice NAR2-like gene,has no known transport activity,but is required to complement high-affinity NO3-transport.Yeast two-hybridisation showed that OsNAR2.1 interacted with OsNRT2.1/2.2/2.3a and knockdown of OsNAR2.1 suppressed expression of the three high-affinity-transport-system NO3-transporters,unlike OsNRT1.1.Knockdown of OsNAR2.1 impaired NO3-uptake and had only-65%of N concentration in wild-type plants under low NO3-supply,while no difference was observed under low NH4+as the sole N source.In this study,the root morphology and auxin distribution were investigated in two osnar2.1 mutant lines and two overxepression lines relative to wild-type(WT)plants under NO3-and NH4+ nutrition at a 0.2 mM N concentration.The results were listed as follows:1.Phytagel Medium experiments were conducted to study the effects of 0.2 mM NO3-and NH4+ on the growth of root in rice at the seedling stage.Compared with WT,the length of seminal and adventitious roots was shorter in RNAi mutants and overexpression lines under NO3-supply.However,lateral root(LR)density decreased by-20%in two RNAi lines and increased by-24%in two overexpression lines under NO3-supply in comparison with WT.No difference was recorded among WT,RNAi mutants and overexpression lines under NH4+ supply.2.Phytagel Medium experiments were conducted to study developmental process of LR primordium in WT,mutants and overexpression lines under 0.2 mM NO3-and VH4+.Compared with WT rice plants,lower unmerged and merged LR primodia and LR in seminal root was observed in two RNAi mutants and higher in two overexoression lines under 0.2 mM NO3-supply.However,no difference was recorded among WT,RNAi mutants and overexpression lines under under 0.2 mM Nh4+.3.Hydroponic experiments were conducted to study whether auxin participated LR formation regulated by low NO3-supply.Compared with WT rice plants,auxin concertration in the root decreased in two RNAi mutants while increased in two overexpression lines.Conversely,auxin concertration in first leaf increased in two RNAi mutants and decreased in two overexpression lines under 0.2 mM NO3-supply.No difference was recorded in auxin distrition among WT,RNAi mutants and overexpression lines.Exogenous NAA was applied to confirm altered auxin concentration in the roots contribute to LR formation in OsNAR2.1 Knockdown and overexpression lines under NO3-supply.Aapplication of 1 and 2 nM NAA induced the density of LR in seminal root in WT,RNAi mutants and overexpression lines.Furthermore,application of 1 nM NAA induced LR density and pDR5::GUS expression in the root tip of osnar2.1 mutants to the similar level as those in WT.Similarly,1 nM NAA induced LR density and pDR5::GUS expression in the root tip of WT to the similar level as those in two overexpression lines.These results confirmed that altered auxin distribution in the two OsNAR2.1 knockdown and overexpression lines play important role in LR formation.4.[3H]IAA transport from the node-base to the root was analysed whether altered auxin distribution in OsNAR2.1 overexpression lines under 0.2 mM NO3-was due to auxin polar transport.Two overexpression lines had higher[H]IAA activity in the root in comparison with WT rice plants.qPCR showed lower expressional level of five PIN genes in the root of two mutant lines and higher expressional level of five PIN genes in the root of two overexpression lines.The expression of OsPIN1c,OsPIN5a-b and OsPIN10a-b might play the important role in auxin polar transport regulated by OsNAR2.1 under NO3-supply.In conclusion,OsNAR2.1 may have a key function in coordinating LR formation via positively regulating auxin transport from shoot to root.