Mechanism Analysis Of NtHB6 Regulation Of Glutamine And Aromatic Amino Acid Biosynthesis In Tobacco Leaves

Tobacco(Nicotiana tabacum L.)is an important foliar economic crop used for its leaves,and the nitrogen is a key factor affecting the yield and quality of tobacco leaves.As one of the main nitrogen-containing compounds,amino acids not only have important physiological functions,but also are closely related to the quality of tobacco leaves.Glutamine and aromatic amino acids participated in the Maillard reaction with high activity,and the corresponding Amadori compounds in the freshly-cured tobacco leaves were also high,which contributed greatly to the aroma production of Maillard during the curing process.In our previous study,based on RNA sequencing results,NtHB6 was selected as an candidate gene which is involved in nitrogen distribution flow in tobacco leaves.When compared with the control plants,the up-regulated genes in NtHB6 overexpressing plants were enriched to multiple processes of nitrogen metabolite synthesis,transport and allocation;and the down-regulated genes were also involved in the regulation of nitrogen metabolite to a certain extent.Metabolome analysis showed that the content of glutamine in NtHB6-overexpressing plants was significantly higher than of control.These results indicated that NtHB6 might be involved in regulation of the metabolic process of glutamine and aromatic amino acids.In this study,the previously obtained NtHB6 overexpression,RNAi and CRISPR plants were grown in the plant growth chamber and experimental field,and their phenotypes were identified,and the quality of tobacco leaves of these transgenic plants were determined.The regulatory network of NtHB6 was explored through RNA-seq analysis on the transgenic plants,combined with DNA Affinity Purification sequencing(DAP-seq),the downstream target genes of NtHB6 were screened and verified.The main results were as follows:1.Analysis of transcriptional activation activity and gene expression pattern of NtHB6The full-length NtHB6 has transcriptional activation activity,and its Nterminal(containing DNA-binding domain)and C-terminal structures also showed transcriptional activation activity.The expression of NtHB6 could be detected in the main organs of K326,and was high in flowers and leaves,indicating that the expression of NtHB6 was tissue-specific.It was also found that the gene sequences of NtHB6 were completely consistent in different tobacco varieties,indicating its sequence conservation.2.NtHB6 regulates the content of glutamine and phenylalanine and affects the quality of leaves in tobaccoThe analysis on the content of nitrogen-containing compounds in leaves of NtHB6 transgenic plants in the field showed that the content of glutamine and phenylalanine in leaves of NtHB6 overexpressing plants was higher or significantly higher than that of control.The content of glutamine in the NtHB6 interference plants were significantly lower than that of control,indicating that the expression of NtHB6 affect amino acid contents.The content of ammonium ion in the interference and gene editing plants was significantly higher than that in the control,and the accumulation of ammonium ion was not conducive to the growth of tobacco leaves.The chemical composition analysis of tobacco leaves showed that the nicotine content,total sugar content and potassium content of NtHB6 overexpressing plants were significantly lower than that of control,interference and gene editing plants;but the total sugar content of NtHB6 overexpressing plants was14.18%,which was close to the optimal value of 15% of the total sugar content of tobacco.These results indicated that NtHB6 might improve the quality of tobacco leaves by positively regulating the content of glutamine and phenylalanine,and reducing the content of nicotine,total sugar and potassium.3.Screening and verification of downstream target genes of NtHB6 in tobacco.Motif enrichment analysis in DAP-seq showed that NtHB6 binds to [CAATAATTG]and [AATNATT] motifs.Combined with the RNA-seq analysis on NtHB6 transgenic plants and DAP-seq results,10 candidate downstream target genes of NtHB6 were screened,including 5 amino acid metabolism pathways,2 hormone regulation pathways,and 3 flowering pathways-related genes.Yeast one-hybrid results showed that NtHB6 binds promoters of the glutamate synthase encoding gene Nt GS2-1,the growth regulator Nt GRF1,the glutamine transaminase encoding genes Nt GA1,Nt GA2 and the xyloglucan endotransglycosidase encoding genes Nt XTH1,Nt XTH1-1.Based on the expression patterns of target genes in the NtHB6 transgenic plants revealed by RNA-seq and q RTPCR,NtHB6 might positively regulates Nt GS2-1 to increase glutamine content in tobacco leaves;positively regulates Nt GA1 to promote spermidine metabolism,which is beneficial to the growth of pollen tubes,therefore lead to early flowering of NtHB6 overexpressing plants;Our previous study showed the length of the capsule in the NtHB6 overexpressing plants increased significantly compared with the control(Li Shixing,2020),which may also be caused by the regulation of Nt XTH expression by NtHB6.In summary,NtHB6 regulated quality of tobacco leaves from two aspects: on the one hand,positively regulating the content of glutamine and phenylalanine,which provides a large amount of highly reactive amino acids for Maillard reaction;on the other hand,it reduces the content of nicotine,total sugar and potassium,thereby improving the quality of tobacco leaves.