| Rice(Oryza sativa L.) is not only a major staple food crop for a large part of the world’s population but also an important model plant for monocot species, as it attracted the attention of researchers due to its small genome size and complete genome sequence. Nitrogen (N) is considered as an important limited factor of crop yields. Both ammonium and nitrate are the major sources of soil N for plants, however, ammonium is the predominant form of N in a paddy soils. Some physiological experiments have shown that lowland rice was exceptionally efficient at acquiring NO3- formed by nitrification in the rhizosphere, and nitrate was main source of N in soil at later growth and developmental stages of rice under the intermittent irrigation. It has been predicted that up to 40% of the total N taken up by rice roots grown under wetland conditions might be in the form of nitrate and the rate of nitrate uptake can be comparable with that of ammonium. But there are few reports about the physiological and molecular mechanisms of how rice take up nitrate. It has already been found that some high affinity nitrate transporters belonging to the NRT2 family in Arabidopsis and Barley need a partner protein called NAR2 for their function. So based on the NAR2 sequences of Arabidopsis and Barley, the homologous gene OsNAR2.1 was found in rice genome database and expression patterns of OsNAR2s and OsNRT2s was analyzed by semi-quantitative RT-PCR. Knockdown of OsNAR2.1 by RNA interference and the RNAi efficiency of OsNAR2.1 was analyzed by semi-quantitative, quantitative RT-PCR and western blot. The nitrogen uptake of osnar2.1 knockdown mutant was analyzed by 15N labeled NH4+ and NO3-. The electro-physiological measurements of osnar2.1 knockdown mutant for root cell in response to nitrate and rice two-component nitrate uptake system was investigated by oocyte heterologous expression system. Further more, the root morphology and yields of osnar2.1 knockdown mutant under different nitrogen form and the potential genes directly or indirectly regulated by OsNAR2.1 were analyzed by gene chip. The physiological and molecular mechanisms of rice nitrate uptake affected by OsNAR2.1 and other possible functions would be understood in this paper. The main results were as follows:1) Expression patterns of OsNAR2s and OsNRT2s was analyzed by semi-quantitative RT-PCR. The expression of OsNAR2.1, OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.4 were immediately induced by and suppressed by ammonium and Asp, but the expression of OsNAR2.2 and OsNRT2.3b was not affected by nitrogen form and four amino acids (Glu, Gln, Asp and Asn). The expression of OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.4 were also suppressed Gln and Asn, but the expression of OsNAR2.1 was not affected. Different concentrations of nitrate had no effects on the expression of all members of OsNAR2s and OsNRT2s. In addition that OsNRT2.3b and OsNRT2.4 were expressed in leaf, and the expression of OsNRT2.4 was stronger in leaf than in root, All other genes were mainly expressed in root.2) Xenopus oocyte heterologous expression shown that OsNAR2.1 not only interacted with OsNRT2.1/OsNRT2.2 (both have the same ORF), but also with OsNRT2.3a to give functional nitrate uptake, and its homologous gene OsNAR2.2 could not replace it for function. Interestingly OsNRT2.3b which only had 30 amino acids difference from OsNRT2.3a alone couldn’t transport nitrate. The Km value for nitrate uptake was 0.03 mM for OsNRT2.1/OsNRT2.2-OsNAR2.1 and 0.34 mM for OsNRT2.3a-OsNAR2.1.3) Two lines of OsNAR2.1 highly silenced mutants by RNAi were obtained. The silence efficiency of them analyzed by quantitative RT-PCR were 84.6% for rl 79.1% for r2. Using OsNAR2.1 specific antibody for Western blot analysis shown that the expression of OsNAR2.1 was also lower in osnar2.1 knockdown mutant than in WT. Interestingly knockdown of OsNAR2.1 by RNA interference synchronously suppressed expression of three members of the NRT2 nitrate transporter gene family (OsNRT2.1, OsNRT2.2 and OsNRT2.3a), but had no effects on the expression of OsNAR2.2, OsNRT2.3b, OsNRT2.4 and OsNRTl.1 in the mutants.4) The nitrogen uptake of osnar2.1 knockdown mutant analyzed by 15N labeled NHV+ and NO3- shown that their nitrate uptake system was impaired greatly under both low and high concentrations of external nitrate supply, but ammonium uptake system was not affected. Compared to the WT plants, both rl and r2 mutants had decreased nitrate influx rate into roots by 84-86% at 0.2 mM 15N-NO3-and by 69-73% at 5 mM 15N-NO3-. The electro-physiological measurement of nitrate-elicit changes in root cell membrane potential also showed that both low- and high- affinity nitrate uptake system was impaired in the osnar2.1 knockdown mutants.5) Young seedlings of two osnar2.1 knockdown mutants and WT were grown in 0.2 mM NO3- as the sole N supply, the osnar2.1 knockdown mutants were much smaller than WT plants and the leaf yellowing was also observed. The biomass and total N concentration of two osnar2.1 knockdown mutants root and shoot were lower than that of WT, and these significant differences between mutants and WT were also found even if under 5 mM NO3- supplied. But there was no significant difference between mutants and WT under the condition of 0.2 mM NH4+. These results were confirmed by seedlings treated with nitrogen form and two kinds of nitrate concentration at later growth stages, in addition to that the seed setting and yields of two osnar2.1 knockdown mutants were much lower than that of WT.6) The effects of OsNAR2.1 knockdown on rice root morphology. Main roots of two osnar2.1 knockdown mutants had no significant difference from that of WT, but lateral root length of mutants was shorter than that of WT when seedlings grown on vertical plates containing 0.2 mM NO3-. Under the condition of 0.2 mM NH4+, there were no significant difference of root morphology between mutants and WT. Further it was found that the expression of OsMADS homologous gene ANR1 which was involving in the signal pathway of lateral root development was suppressed in osnar2.1 knockdown mutants and induced by nitrate. So it’s presumed that the effects of OsNAR2.1 knockdown on lateral root growth is due to expression of OsMADS suppressed.7) The results of the whole-genome expression of osnar2.1 knockdown mutants under 0.2 mM NO3- supply analyzed by gene chip confirmed that nitrate uptake system was impaired by OsNAR2.1 knockdown, in addition that oxygen transport was also suppressed. Further analysis of the expression of OsPAD4 homologous gene AtPAD4 which was involving in aerenchyma formation, was suppressed in osnar2.1 knockdown mutants and induced by nitrate.Taken together, rice nitrate transport accessory protein gene OsNAR2.1 was not only required for OsNRT2.1/OsNRT2.2 and OsNRT2.3a for function, but also may directly or indirectly regulate the expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a. The effects of OsNAR2.1 knockdown on both high and low concentration of nitrate uptake. In addition to that OsNAR2.1 knockdown affected lateral root growth possibly by suppressing the expression of OsMADS. |
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