The Mechanisms Of MiR827-NLA-NRT1.7 Circuit Regulates Source-to-Sink Remobilization Of Nitrate In Arabidopsis

Nitrogen remobilization is one of the key steps to improve nitrogen use efficiency. Nitrate remobilization among different tissues was mainly mediated by nitrate transporters(NRT). the regulation patterns of these NRTs are still lacking. At-NLA(Nitrate Limitation Adaptation) was identified from a mutant RING domain truncated that failed to develop essential adaptive responses to 3 mM KNO3 nitrate limitation. Further researches showed that the early senescence in nla mutants under N-deficient condition was due to Pi toxicity, but the relationship between NLA and nitrate, the functional significance of NLA in adaptive responses to sever/mild nitrate limitation remains to be explored. This study aims to explain the phenomenon of hypersensitivity to nitrate limitation in nla mutant using hydroponics, nitrogen analysis, 15N-nitrate tracing and uptake experiments. Finally, elucidate the mechanism of NLA and its target protein that participates in nitrate remobilization, and indicates the functional significance of NLA in adaptive responses to sever/mild nitrate limitation. The specific results are as follows:1 Under nutrient-replete conditions, no growth difference was observed between WT and nla mutants. However, when plants were transferred to the N-deficient medium for 3 days, 95% of nla rosettes showed the typical N-deficient phenotype with discoloration of older leaves contrast to 10% of WT.2 N-deficiency for 3 days significantly reduced the total N concentrations of both WT and nla plants, which were lower than those of corresponding plants under N-replete conditions. Under N-deficient conditions, the total N concentrations in older(first two pairs) and younger(other pairs) leaves of WT reduced by 15.5% and 24.3%, respectively; while in nla mutant, N-limitation caused a much more obvious decrease in the total N concentrations in older leaves than in younger ones. These results further suggested that NLA was involved in N-remobilization from old leaves to new leaves in Arabidopsis.3 To further confirm the result of 2, we arranged 15N-nitrate tracing and uptake experiments. 15N-nitrate was spotted on the first two pairs of rosette, and after 24 h, 15 N concentration in the younger leaves of nla mutant was ~11%, which was about 2 times as much as that of WT. Consistent with 15N-nitrate tracing results, 15 N concentration in the younger leaves of nla mutant was also significantly higher than that of WT, and the younger leaves/older leaves partition of 15 N was 7.57 in nla mutant but only 4.08 in WT.4 NRT1.7 mediated phloem loading of nitrate in source leaves to remobilize nitrate from older leaves to new leaves. Using Ubpred software online, we found there were three high-score ubiquitination sites, two at the N-end(Lys-21 and Lys-34) and one Lys residues(Lys-606) located in the central loop of NRT1.7.5 NMY51 yeast that contained pPR3-NLASPX and pBT3-NRT1.7 vectors could grow normally on SD-His-Ade-Trp-Leu medium. The interaction between NLASPX and NRT1.7 activated β-galactosidase therefore yeast turned blue. Reconstituted yellow fluorescent protein(YFP) signals from co-expression of nYFP-NLA and cYFP-NRT1.7 were observed in the plasma membrance. Also, Co-IP and Pull-down assays further found the interaction between NLA and NRT1.7.6 In western blot assay, evidences of down-regulation of NRT1.7 protein abundance in 35S::NLA/35S::Myc-NRT1.7 double transgenic plants compared to 35S::Myc-NRT1.7 transgenic plants, and up-regulation of NRT1.7 protein abundance in nla mutants compared to WT suggested NRT1.7 could be degraded by NLA. In vivo ubiquitination assay, NRT1.7 could be ubiqintin by NLA but not NLAmut.7 After nitrate deprived, miRNA827 accelerated dramatically, while NLA transcripts didn’t decrease and NLA protein declined dramatically in WT plants. Under nitrate-limitation conditions, NLA protein didn’t change in mir827, dcl3 and rdr2 mutants compared with WT. These results indicated that translational repression of NLA was dependent on miR827.8 miR827 was blocked in biogenesis mutants, dcl3 and rdr2. Compared to WT, the levels of NLA in these two mutants did not increase even though miR827 was absent. However, the NLA protein abundance was much higher in these two mutants than in WT. We also detected NLA protein abundance in mir827 knock-out mutant. The results were consistent with the results obtained with dcl3 and rdr2. Overexpression MIR827 down-regulated NLA protein accumulation and did not significantly affect NLA transcripts. These suggested miR827 repressed NLA at translational levels.Thus, our findings discover an important mechanism that plants regulate source-to-sink remobilization of nitrate through the integration of miRNA-guided translational repression and ubiquitin-mediated posttranslational regulatory pathway.