Study On The Mechanism Of Action Of Tomato SlGRF4 To Improve Nitrogen Utilization Under Low Nitrogen Stress

Nitrogen is one of the essential nutrients for plant growth and development.In agriculture,nitrogen fertilizers are often applied to ensure an adequate supply of nitrogen for crop production.However,excessive use of nitrogen fertilizers can affect normal plant growth and cause problems such as soil salinization and compaction.Growth-regulating factors（GRFs）play an important role in regulating plant growth and development.This study investigates the mechanism of action of SlGRF4 in tomato（Solanum lycopersicum）and the function of overexpressing SlGRF4transgenic tomato under low nitrogen conditions.The following results were obtained:1.Low nitrogen stress（0.2 m M）inhibited the growth of tomato seedlings,decreased chlorophyll content,and significantly reduced the content of nitrate nitrogen in the plant.Transcriptome analysis revealed 686 differentially expressed genes after low nitrogen stress,with 499 up-regulated genes and 187 down-regulated genes.The expression levels of four Sl GRF family members（Sl GRF1L,Sl GRF3,SlGRF4,and SlGRF4L）were significantly down-regulated.Gene enrichment analysis showed that the functions of differentially expressed genes were mainly enriched in stress response and nitrogen metabolism regulation,while metabolic pathways were mainly enriched in nitrogen metabolism and amino sugar and nucleotide sugar metabolism.Nitrate transporter,carbonic anhydrase,and glutamate dehydrogenase in nitrogen metabolism pathway were significantly up-regulated,while nitrate transporter,nitrite reductase,carbonic anhydrase,glutamate dehydrogenase,glutamine synthetase,and glutamate synthase were significantly down-regulated2.Phylogenetic analysis of SlGRF4 revealed that the Sl GRF family was divided into four subfamilies,with SlGRF4 belonging to Class III.Sequence alignment revealed that GRFs contain highly conserved QLQ and WRC domains.Gene structure and promoter analysis of SlGRF4 showed that it has a very large intron sequence,and its promoter sequence contains binding sites for hormones,light-responsive elements,as well as MYB and MYC transcription factors.3.The yeast two-hybrid experiment revealed that SlGRF4 can interact with the Sl GIF family members Sl GIF1,Sl GIF2L,and Sl GIF3.SlGRF4 protein was successfully induced by prokaryotic expression,and S-nitrosylation modification was found to occur on this protein.EMSA experiments demonstrated that SlGRF4 can bind to the promoter of Sl KNOX2 and regulate its expression.4.Transgenic tomato plants overexpressing SlGRF4 were successfully obtained,and their functions under low nitrogen conditions were analyzed.Under low nitrogen stress,the root length and fresh weight of seedlings overexpressing SlGRF4 were significantly increased compared to the wild type（WT）.After 15 days of treatment with low nitrogen（0.2 m M）,the H₂O₂ and MDA contents in the leaves of transgenic plants were lower than those in WT,while the activities of antioxidant enzymes POD and CAT,the content of chlorophyll a,the content of nitrate,the expression of key enzymes（Sl NR,Sl GS,and Sl GOGAT）in nitrogen metabolism pathways,and the enzyme activities of NR and GS were higher than those in WT.Transcriptome analysis after low nitrogen treatment revealed 824 differentially expressed genes in transgenic plants compared to WT,including 340 upregulated and 484 downregulated genes.GO functional enrichment analysis indicated that more differentially expressed genes in transgenic plants were enriched in oxidoreductase activity.KEGG pathway enrichment analysis showed that the overexpressing plants mainly enriched in amino acid metabolism pathways.Further analysis of nitrogen metabolism pathways revealed that the expression of all four key enzymes（Sl NR、Sl Ni R、Sl GS、Sl GOGAT）was significantly upregulated.Analysis of differentially expressed transcription factors showed that there were 258 differentially expressed transcription factors in the overexpressing plants after low nitrogen treatment,including 169upregulated and 89 downregulated genes.The b HLH family was the most abundant family of differentially expressed transcription factors,with 29 members,of which 20were upregulated and 9 were downregulated.