Effects Of Nitrogen Supply And Senescence On Iron And Zinc Translocation And Accumulation In Grain Of Winter Wheat

Malnutrition has been a global hot-spot problem since over half of the global population is suffering the zinc (Zn), iron (Fe) and vitamin deficiencies. Wheat is the dominant staple food comprised over50%of diet and micronutrients content in the grain and are also the main source for human micronutrients absorption. Nitrogen (N) application has been approved to be an effective practice on Fe and Zn accumulation in wheat grain. Less research has been published on how nitrogen application affects the Fe and Zn uptake and accumulation in wheat and the further mechanism between N supply and nitrogenous chelator related to Fe and Zn uptake and translocation.During the monocarpic senescence of cereals plant like winter wheat, positional cloning of GPC-B1genes showed GPC-B1is a NAC domain transcription factor related to senescence acceleration and grain protein and micronutrients remobilization in wheat which resulted in high grain protein and micronutrients accumulation in wheat grain. In this study, the effects of N application on nicotianamine synthase (NAS) genes expression, Fe and Zn uptake and translocation were conducted under potting and hydroponic culture condition. Meanwhile, we identified function of the orthologous genes of GPC-1and evaluated its effects on senescence, Fe and Zn remobilization processes and candidate genes in wheat through different GPC-1genotype mutants. And the RNA seq between wild type and GPC knockout mutants at three stages of senescence was applied to determine the underlying transcriptional changes associated with these phenotypic traits.The main results are as followed:1. Under potting culture condition, N application enhanced leaves dry weight and their Fe and Zn content; meanwhile, root dry weight and their Fe and Zn content were decreased with increased N application. With increased N supply, the expressions of TaNAS2and TaNAS3in leaves were improved with the nitrogen supply in leaves and the expression of TaNASl was specifically up-regulated in roots. These results indicated that increased N supply enhanced NAS gene expression, which may potentially result in more nicotianamine concentration in roots and/or leaves;2. Under hydroponic culture condition, leaves dry weight and their Fe and Zn content were increased and roots dry weight and their Fe and Zn content were decreased with the increased N level. With increased N levels, TaNAS2expression in leaves was highly up-regulated with increased N supply and the expression of TaNASl in root was up-regulated. The similar NAS genes expression pattern in wheat grown in potting and hydroponic culture, exhibited that the enhancement of NAS genes expression had highly responses to increased N supply.3. The gpc-1mutants were used to identify the effect of GPC-1on grain protein, Fe and Zn remobilization. The results showed that a significant senescence delay in both gpc-al and gpc-dl single mutants and a stronger effect was found in gpc-1double mutant than in the wild type. At maturity, grain protein concentration, Fe and Zn concentrations in all gpc mutants were significantly lower than in wild type while thousand grain weight was unaffected. Seven target genes showed significant differences in expression between gpc-1double mutant and control plants at three different stages. GPC-A1and GPC-D1homoeologues played redundant roles in senescence and nutrient remobilization processes;4. A significant delay in senescence was found in gpc-al single mutant compared with wild type. This result also confirmed that the GPC-1gene played an important role in the initiation and progression of senescence. Since the gpc-al/b2double mutant showed similar senescence traits as gpc-al single mutant, GPC-B2was predicated to play non-functional role in tetraploid wheat. Based on the mRNA sequence analysis and phenotype observed in the field experiment, GPC-A2may have function in senescence process regulation in wheat;5. Under the potting culture condition, RNA sequence analysis was used to compare the difference in the transcriptome of wheat during the senescence at heading stage,12days and22days after anthesis. The results showed that the genes expression related to catalytic enzyme and transporter were down-regulated at heading stage and12days after anthesis. With the onset of wheat senescence, the genes expression related to hydrolase enzyme were highly up-regulated at22days after anthesis, which indicated that grain N accumulation may partly come from protein degradation in flag leaves at grain filling stage;6. Based on transcriptome analysis of GPC-RNAi transgenetic plant, isogroup08662and isogroup10136as plant hormone were up-regulated by GPC. By using TILLING for GPC4XDNA pool generated by tetraploid wheat (Desert durum(?)heat, Kronos),5potential target mutants of isogroup08662were identified. The sequencing results showed that synonymous mutation and systemic error existed in isogroup08662. Among three homologues of isogroup10136, isogroup10136-2belonged to wheat D genome. And23potential mutants of isogroup10136-3were prepared for the precondition for phenotype and gene function characterization.