| As a representative fruit tree of the Rosaceae family,apple is widely grown in northern China.The level of sugar and acid in the fruit is closely related to the taste of the fruit and is an important indicator of fruit merchantability.And the type and composition of starch and soluble sugar greatly affect the quality of apples.At the same time,fruit trees have different uptake and utilization efficiency of nitrogen nutrition in different periods.In actual production management,the application of nitrogen should be reasonably regulated in stages according to the growth characteristics,nitrogen demand characteristics and storage nitrogen nutrition characteristics of fruit trees in different periods,combined with the principle of high yield and excellent production.At present,most of the studies on the effects of nitrogen on carbon and nitrogen metabolism and fruit quality are at the physiological level,but the molecular mechanism of how transcription factors in apples respond to nitrogen to regulate the expression level of genes related to sugar metabolism and thus regulate apple sugar metabolism is still unknown.Therefore,in this study,apple fruit and callus were used as research materials to explore the mechanism of Mdb ZIP44 regulating sugar metabolism.The main results were as follows:1.Soluble sugar content and organic acid content were analyzed at 30,60,90,120,135and 150 days after full bloom（DAFB）in‘Oregon Spur 2’apples treated with different nitrogen levels(0,150,300 and 600 kg·hm-2).The results showed that nitrogen fertilization at 150,300 and 600 kg·hm-2 significantly increased the fructose content at 30 DAFB;150,300 and 600 kg·hm-2 significantly increased the glucose content at 120 DAFB;300 kg·hm-2 significantly increased the sorbitol content at 90,120 and 150 DAFB;150 kg·hm-2significantly increased the sorbitol content at 150 DAFB.Nitrogen fertilization at 150kg·hm-2 showed the highest sucrose content and was significantly higher than the control.Nitrogen fertilization at 150,300 and 600 kg·hm-2 significantly reduced malic and quinic acid content at 30 DAFB;Nitrogen fertilization at 150,300 and 600 kg·hm-2 significantly reduced citric acid content at 60 and 120 DAFB;Nitrogen fertilization at 600 kg·hm-2significantly reduced ascorbic acid content at 60,120 and 150 DAFB.2.Transcriptome sequencing analysis was performed on apple fruits at different periods and treatments(60,120 and 150 DAFB,0,300 and 600 kg·hm-2 N fertilizer treatments).The results showed that the differentially expressed genes（DEGs）were mainly enriched in glycolysis,phenylpropanoid synthesis and phytohormone signaling pathways at60 DAFB;the DEGs were mainly enriched in cell phagocytosis,photosynthetic organ carbon fixation and photosynthesis pathways at 120 DAFB;the DEGs were mainly enriched in flavonoid metabolism,carotenoid synthesis,pathogenic bacteria interactions pathways at150 DAFB.sugar and acid signaling pathways showed that 27 DEGs were involved in starch and sucrose metabolism and glycolysis pathways,mainly including Mdα-GP,Md SPS,Md SUS,Md FRK Md PFK,Md FBA,etc.;8 DEGs were involved in pyruvate metabolism pathway,mainly including Md PK,Md DLD,Md DLAT,Md PPC,Md MDH,etc.The q RT-PCR results showed that the expression of DEGs related to sugar and acid metabolism were consistent with the expression pattern of transcriptome data.3.The transcriptome was screened for Mdb ZIP44 in response to nitrogen induction,in order to further explore whether it regulates starch and soluble sugar accumulation.Mdb ZIP44 overexpression,knockout and silencing vectors were constructed.Mdb ZIP44overexpression tomato,Mdb ZIP44 overexpression and knockout apple callus were successfully obtained through Agrobacterium-mediated genetic transformation.The results showed that the content of starch and sucrose in tomato and apple callus with Mdb ZIP44overexpression was significantly higher than that in wild type（WT）plants when treated with nitrogen,while the glucose content was significantly lower than that in WT lines.The GP enzyme activity of tomato and apple callus overexpressed with Mdb ZIP44 was significantly lower than that of WT lines,the SPS enzyme activity was significantly higher than that of WT lines,and the expression of genes related to sugar and acid metabolism was also significantly higher than that of WT lines,while the effect of Mdb ZIP44 gene knockout on apple callus was opposite.4.Mdb ZIP44 silence and Mdα-GP2 overexpression vectors were transiently injected into apple fruits,and Mdα-GP2 overexpression had no effect on the expression level of Mdb ZIP44 gene when Mdb ZIP44 was repressed,when Mα-GP2 was overexpressed,the inhibition of Mdb ZIP44 would activate the expression of Mα-GP2.The results suggested that Mdb ZIP44 further regulates sugar metabolism in apple fruits by regulating the expression of Mdα-GP2 gene.The analysis of starch and soluble sugar content showed that inhibition of Mdb ZIP44 gene expression or overexpression of Mdα-GP2 gene decreased starch content and increased glucose content,and inhibition of Mdb ZIP44 gene and overexpression of Mdα-GP2 gene co-transformed apple fruits decreased starch content and increased glucose content to a greater extent.5.Three G-box elements were detected in the first 2000 bp of Mdα-GP2 gene promoter.Yeast one-hybrid（Y1H）and electrophoretic mobility shift assay（EMSA）experiments confirmed that Mdb ZIP44 directly binds to the promoter of the Mdα-GP2 gene and interacts with all three G-box elements.Dual-Luciferase（LUC）analysis showed that Mdb ZIP44negatively regulates the expression of Mdα-GP2 gene.The interaction of Mdb ZIP44 protein with Md CPRF2-like protein was demonstrated by yeast two-hybrid（Y2H）,bimolecular fluorescence complementation（Bi FC）and pull down assays,and Md CPRF2-like transcription factor is also a member of the apple b ZIP family.Moreover,Md CPRF2-like and Mdb ZIP44 combined with Md CPRF2-like can positively promote the expression of Mdα-GP2 gene.This study have resolved the molecular mechanism of Mdb ZIP44 transcription factor and key genes in response to nitrogen and thus regulate apple sugar metabolism through Mdb ZIP44-Md CPRF2-like-Mdα-GP2 regulatory module,which provides a new idea for quality regulation in horticultural crops.These findings not only help us to understand the regulatory network of apple sugar metabolism,but also have important implications for enhancing fruit breeding and improving fruit quality. |
