Functional Study Of NP-A Subclass Genes In Response To High Ammonium In Populus Trichocarpa

Nitrogen is the main component of nucleic acid,protein,and chlorophyll in plants,and plays a crucial role in plant growth and development.Inorganic nitrogen sources absorbed by plants from soil mainly include NO₃^-and NH₄⁺.Under waterlogged,low temperature,and acidic conditions,NH₄⁺becomes the main nitrogen source used by plants.Excessive ammonium can have a toxic effect on plants,severely inhibit plant growth.Researches have shown that pathways such as nitrogen absorption,nitrogen assimilation,cation signaling and carbon nitrogen metabolism play important roles in the alleviation of plant ammonium toxicity.However,the complete regulatory mechanism has not been clarified,and whether other key regulatory pathways are involved remains to be studied.This study explored the response genes of Populus trichocarpa under high ammonium conditions through transcriptomic strategies,and conducted functional studies on several nucleoside phosphorylase（NP）family genes in response to high ammonium through genetic,physiological and biochemical analysis,wood anatomy,and transcriptome analysis.The main results are as follows:（1）Studies on the growth of wild type P.trichocarpa under different nitrogen forms and concentrations showed that high concentrations of ammonium significantly inhibited plants growth compared to mixed forms of ammonium and nitrate,or nitrate nitrogen.Transcriptome analysis showed that in addition to the up-regulated expression of key genes for ammonium assimilation such as glutamine synthetase and glutamate dehydrogenase,several genes involved in the nucleoside metabolism pathway were also significantly up-regulated（Potri.013G100700,Potri.013G100800,Potri.013G1010000）in the barks and leaves of P.trichocarpa under high ammonium stress,suggesting that they may play an important role in the response to high ammonium in P.trichocarpa.（2）Identified NPs genes responsive to high ammonium.The genome database search of P.trichocarpa revealed that above three significantly upregulated genes belong to the NPs family genes.There are 14 NPs in P.trichocarpa,which are divided into four subclasses:A,B,C,and D.The results of tissue expression and promoter driven GUS reporter gene expression showed that NP-A subclass genes（PtNP10,PtNP11,PtNP12）were highly expressed in phloem and cambium,and were strongly induced by high ammonium.PtNP10 and PtNP12 are located in the cytoplasm.（3）The NP-A subclass genes are involved in altering the NH₄⁺assimilation ability of P.trichocarpa with redundant functions.Based on CRISPR/Cas9 gene editing technology,single,double,and triple genes editing mutant lines of np12,np10np11,and np10np11np12 were created.Under normal ammonium condition,knockout of NP-A subclass genes enhanced the NH₄⁺absorption and assimilation ability of young trees,but did not affect plant growth.Under high ammonium condition,knockout of the NP-A subclass genes enhanced the NH₄⁺assimilation ability of the aboveground tissues of young trees,making the mutants exhibit significant high ammonium tolerance characteristics.（4）We created OE-PtNP10 and OE-PtNP12 overexpression lines and found that PtNP10plays a major role in altering the NH₄⁺absorption and assimilation ability.Under normal ammonium condition,overexpression of PtNP10 gene decreased the NH₄⁺absorption and assimilation capacity of young trees,increased the number of stem nodes,and decreased the length of internodes.Under high ammonium condition,overexpression of PtNP10 gene decreased the ability of NH₄⁺assimilation of aboveground tissues,leading to a significant accumulation of NH₄⁺,making OE-PtNP10 overexpressed young trees significantly sensitive to high ammonium.Under two nitrogen supply levels,overexpression of PtNP12 gene did not change the NH₄⁺uptake and assimilation ability of young trees.（5）PtNP10 plays an important role in regulating vascular cambium activity and wood tissue formation.The results of phenotypic analysis of plants growth in soil habitats showed that the knockout of NP-A subclass genes,and overexpression of PtNP12 gene did not affect the growth of P.trichocarpa,while overexpression of PtNP10 gene resulted in multiple phenotypic changes,including shortening of internodes,increasing the number of stem nodes,promoting axillary bud growth,and reducing leaf size.Microstructural analysis showed that overexpression of PtNP10 gene significantly reduced xylem width,xylem cell layers,cambium layers,xylem fiber and vessel cell sizes,and secondary wall thickness.The results of wood component analysis showed that overexpression of PtNP10 or PtNP12 gene could reduce lignin and cellulose content.Compared with WT,there were no significant differences in stem microstructure and wood components among various mutants.（6）Transcriptome sequencing was performed on the barks and leaves of WT,np10np11np12-3 mutant and OE-PtNP10-8 overexpression plants treated with normal and high ammonium,respectively.A large number of differentially expressed genes（DEGs）were obtained among the comparative groups,and DEGs were significantly enriched in the auxin activation signal pathway,glutathione metabolism pathway,chitin response,and phloem development process.Genes expression analysis revealed that NP-A subclass genes not only participate in nitrogen assimilation under high ammonium stress,but also can respond to high ammonium stress by altering auxin activated signal transduction,stress response,and the expression of P-proteins in the phloem.In summary,there are 14 NPs in P.trichocarpa,and the expression of NP-A subclass genes are strongly induced by high ammonium.Knocking out NP-A subclass genes enhance the NH₄⁺assimilation ability of aboveground tissues,making young trees have high ammonium tolerance characteristics.Overexpression of the PtNP10 reduces the ability of NH₄⁺assimilation of aboveground tissues,resulting in the high ammonium sensitivity of young trees.NP-A subclass genes change nitrogen utilization efficiency to cope with high ammonium stress by integrating auxin activated signal transduction,stress response,phloem development and other signal pathways.This study identified important forest tree genes that adapt to high ammonium habitats,laying the foundation for cultivating new forest germplasm materials with efficient nitrogen utilization in the future.