Study On Nitrogen Form Preference Of Blueberry And Its Related Mechanism

As a‘life element’,nitrogen（N）is not only a component of biological macromolecules such as protein and nucleic acid,but also participates in plant photosynthesis and mineral element absorption.Nitrogen deficient in plants leads to slow growth,narrow and yellow leaves as well as weak root system.The inorganic nitrogen sources used by plants are mainly ammonium nitrogen（NH₄⁺-N）and nitrate nitrogen（NO₃^--N）.Blueberry（Vaccinium spp.）,an economic plant with high nutritional and economic value,is extremely strict in soil quality and has obvious nitrogen inefficiency.Long-term application of ammonium nitrogen fertilizer can reduce cation exchange capacity,basicity saturation and exchangeable base ions,and increase exchangeable acid obviously,which makes soil acidification.Thus,blueberry is suitable for growth in acidic soil and is considered to be a traditional‘ammonium plant’.However,it has been reported that the application of NH₄⁺-N could reduce the pH in soil and increase the content of N,phosphorus（P）,iron（Fe）in blueberry leaves.But when only ammonium nitrogen was applied,the blueberry showed a decline in growth.In addition,the application of a proper proportion of NO₃^--N combined with NH₄⁺-N could improve the quality of blueberry.Some studies even showed that blueberry could use NO₃^--N as the sole nitrogen source to maintain its growth.Therefore,blueberry is not a simple‘ammonium plan’,its absorption preference for different nitrogen forms and related regulation mechanisms are still unclear.In this study,the field fertilization experiment of different nitrogen forms of blueberry was carried out,and the physiological responses of each treatment group were determined.Meanwhile,the expression profiles of key genes involved in nitrogen metabolism were also assayed to investigate the effects of different nitrogen forms on nitrogen metabolism in blueberry.Furthermore,the cDNA length of nitrogen metabolism gene AMT3（Ammonium transporter 3,AMT3）,NRT1.5（Nitrate transporter 1.5,NRT1.5）,NRT2（Nitrate transporter 2,NRT2）,NR（Nitrate reductase,NR）and NiR（Nitrite reductase,NiR）were isolated from blueberry,and the expression vector was constructed and transformed into wild type Arabidopsis thaliana,so as to explore the intrinsic mechanism of nitrogen form preference of blueberry.The specific research results were listed as following:After three months of treatment with different nitrogen forms,there were significant differences in the biomass growth of blueberry plants.The growth of blueberry plants in pH 5.0 treatment group was generally better than that in pH 7.5 treatment group,in which the best growth rate was at 2:1 of ammonium to nitrate ratio.The plant height of each group tended to be the same,but significant difference occurred in crown amplitude.The maximum crown amplitude was observed in the treatment group at 2:1,which was 10.89%more than that of the control.The chlorophyll contents in the mixed nitrogen treatments were higher than that in the single nitrogen form treatments,which meaned mixed nitrogen treatment promoted the absorption of nitrogen and improved the photosynthetic effeciency of the buleberry.Meanwhile,the NR of mixed nitrogen treatment group showed high activity.A positive correlation were observed between NiR activity and NR activity in all treatments,but the NR activity was generally lower than NiR activity,which may be an important reason for the low absorption efficiency of NO₃^--N in blueberry.The results of qRT-PCR showed that the expression of VcAMT3 was up-regulated under mixed nitrogen treatment.With the decrease of the ratio of NH₄⁺-N in mixed nitrogen,the expression of VcAMT3 was exhibited a typical inverted‘V’trend,which increased the absorption efficiency of NH₄⁺-N.However,the transcription level of NRT1.5,NRT2 was significantly increased under the treatment of pH 5.0,which enhanced the utilization of NO₃^--N in blueberry.The highest expression level of the genes related to ammonium and nitrate transport occurred in the treatment group with an ammonium to nitrate ratio of 2:1.The expression of NR,NiR was regulated by the concentration of NO₃^-.The expression of NR,NiR was up-regulated in the pure NO₃^--N treatment group,but rarely in the pure NH₄⁺-N treatment group.Meanwhile,the expression level of NR,NiR was highest when the ratio of ammonium to nitrate was 2:1.This indicated that the gene expression which related to ammonium and nitrate nitrogen transport and nitrate reductase was regulated by exogenous nitrogen ratio and pH,and the up-regulation of gene expression was beneficial to nitrogen metabolism in blueberry.The cDNA of VcAMT3,VcNRT1.5,VcNRT2,VcNR and VcNiR,with the full length of722,2342,765,2670 and 1258 bp,encoded 175,185,255,889 and 419 amino acids respectively.And their respective isoelectric points were 9.16,9.21,6.93,6.19 and 8.70.The phylogenetic tree and amino alignment showed that most of the genes involved in nitrogen metabolism of blueberry had high similarity with Actinidia chinensis.The bootstrap values of NR,NiR gene in Actinidia deliciosa were 93,83,respectively.Furthermore,the results of TMHMM analysis indicated that there might be 5,2 and 5transmembrane structures in AMT3,NRT1.5,NRT2,which belonged to membrane protein,while NR,NiR perhaps have no transmembrane structure and were not transporters.After transferring the key genes into Arabidopsis thaliana,the growth of Arabidopsis thaliana plants with VcNRT2,VcNiR overexpression was superior to that of wild-type plants under normal substrate culture.VcNRT1.5,VcNR could not bloom and fruiting normally at the later stage.After 1 month of coconut bran culture with different nitrogen sources,the overexpressed plants in the treatment group with NO₃^--N as the sole nitrogen source were superior to those in the NH₄⁺-N treatment group and the control group,among which the VcAMT3 overexpressed Arabidopsis thaliana plants had highest biomass growth.The plant height and leaf disc size were 11.24 and 2.05 times of that of wild type plants,respectively.In the NH₄⁺-N treatment,all the plants died in seedling stage except VcNiR overexpression plants.The results showed that VcAMT3,VcNRT2,VcNiR gene played an important role in nitrogen metabolism and could improve the nitrogen absorption ability of Arabidopsis thaliana plants.In conclusion,blueberry prefers the nitrogen form of ammonium nitrate mixture.The ratio of ammonium to nitrate being 2:1 was most suitable for growth of blueberry,indicated by the highest nitrogen utilization efficiency and highest biomass growth of blueberry.Enhancing the activity of NR and NiR to improve the utilization of NO₃^--N in blueberry may be the the key to solve the low nitrogen nutrition efficiency in blueberry.