Functional Study Of Apple MdGBF3 Gene Response To Nitrogen Level In Regulating Flowering And Growth Development

Appropriate nitrogen supply is an important guarantee for flowering and high yield of fruit trees.At present,most of the orchard apple production in our country there is a serious excessive or unreasonable lack of nitrogen fertilizer application,resulting in young trees and adult tree development defects（excessive vegetative growth or tree premature aging and other development problems）,causing difficulty in flowering,poor quality of flower buds,big and small year results and other issues;at the same time,with the gradual northward and eastward expansion of apple high-yield areas in China,fruit trees have encountered more serious nitrogen deficiency or high nitrogen stress conditions caused by abuse of nitrogen fertilizer,which aggravates a series of industrial problems such as growth and development,flowering and fruiting.Therefore,it is of great theoretical and practical significance to clarify the response and utilization of nitrogen in fruit trees and its relationship with flowering,to explore the key regulators of apple nitrogen stress（nitrogen deficiency or high nitrogen environment）to regulate flowering and growth,and to analyze its mechanism of action,so as to improve the utilization rate of nitrogen fertilizer and realize the high quality and high yield of apple industry under different site conditions.In this study,we used the RNA-seq data of nitrogen treatment of different apple rootstock resources and the expression characteristics of nitrogen response materials of apple‘GL3’tissue culture seedlings,and found that a MdGBF3 gene was significantly induced under nitrogen deficiency and high nitrogen supply.High expression,which may be involved in regulating apple flowering and growth and development in response to nitrogen stress（nitrogen deficiency or high nitrogen）;the biological functions of Arabidopsis,poplar and apple transgenic systems were further confirmed.The key interaction protein MdIDD8 and its potential downstream regulatory target genes were explored and identified by yeast two-hybrid,bimolecular fluorescence complementation and DAP-seq.The main results are as follows:（1）Identification of apple MdbZIP transcription factor family and nitrogen response.By aligning the genome of‘Golden Delicious’apple,there are 89 MdbZIP family genes,including8 MdbZIP genes in subfamily G.The candidate MdGBF3 gene was significantly induced by high nitrogen in the roots of M₉-T₃₃₇ and Malus prunifolia Borkh.var.ringo Asami,and the expression of MdGBF3 gene was gradually inhibited when the tissue culture seedlings of apple‘GL3’were transferred from nitrogen-deficient medium to nitrogen-supplied medium,and reached the lowest peak at 12 h.As nitrogen deficiency continued to 7 d,its expression was gradually induced to another peak.In addition,its expression was induced to the highest point when transferred from nitrate nitrogen to nitrogen-deficient medium to day 7.It indicated that MdGBF3 was induced by low nitrogen and high nitrogen.（2）Functional analysis of apple MdGBF3.MdGBF3 gene was located in the nucleus.Under nitrogen deficiency and high nitrogen stress conditions,MdGBF3-OE heterologous overexpression Arabidopsis lines 4#and 7#significantly promoted the growth of roots and shoots under nitrogen deficiency conditions and improved nitrogen utilization.At the same time,MdGBF3 significantly promoted the vegetative growth and early flowering of Arabidopsis under high nitrogen stress.The results showed that MdGBF3 gene had the resistance to nitrogen deficiency and high nitrogen stress and promoted its growth and development,flowering and nitrogen utilization.（3）Screening and identification of MdGBF3 interacting proteins.Yeast two-hybrid and bimolecular fluorescence complementation experiments confirmed that the key interacting protein of MdGBF3 was MdIDD8.The yeast library screening and verification of MdIDD8further confirmed that MdIDD8 interacted with candidate MdGBF3,MdKIN10 and MdHSFB3-LIKE proteins.It indicates that MdGBF3 may interact with candidate key proteins to achieve its biological function.（4）Functional analysis of apple MdGBF3 interacting protein MdIDD8 and screening of its downstream target genes.MdIDD8 is localized in the nucleus.The expression of MdIDD8in apple rootstock roots(M₉-T₃₃₇,Malus sieversii,Malus prunifolia Borkh.var.ringo Asami and Malus baccata)was significantly induced by nitrate nitrogen,and under the condition of nitrogen deficiency and transfer of‘GL3’,it was induced at 0.5 h and reached the first peak at6 h.In addition,MdIDD8-OE heterologous overexpression of poplar phenotype found that MdIDD8 significantly inhibited the growth and development of poplar（plant height,fresh weight of ground and root,root shoot ratio）and leaf quality（leaf fresh weight,length,width and area）,and MdIDD8-OE apple transgenic lines showed developmental defects under normal conditions（poor growth,leaf chlorosis,etc.）.In addition,using DAP-seq technology,five binding motifs of MdIDD8 were found,of which TTTTGTCTTTTT type was the main binding motif,and its potential downstream regulatory target genes were mainly nitrogen and phosphorus transport,auxin and gibberellin signal and anabolic metabolism,sugar signal and anabolic metabolic pathways and multiple transcription factors.It indicated that the interaction protein MdIDD8 of MdGBF3 was involved in the regulation of plant growth and development in response to nitrate nitrogen.In summary,this study found a key MdGBF3 gene induced by nitrogen deficiency and high nitrogen,which interacts with MdIDD8 to participate in the regulation of plant flowering,growth and development and nitrogen utilization.It provides an important theoretical and application basis for solving a series of production problems such as difficult flowering caused by unreasonable site conditions and nitrogen fertilizer application in apple production.