Functional Analysis Of Apple MdAMR1-LIKE In Regulating Ascorbic Acid Biosynthesis

Ascorbic acid（As A）,also known as Vitamin C（Vc）,is an important antioxidant and cofactor of many enzymes in organisms.Fruit is the main natural As A source for humans.The content of As A in fruit mainly depends on the DE novo biosynthesis ability of fruit cells.Compared with kiwifruit and other fruits,the reason of low As A in apple fruit is mainly due to the weak synthesis ability of the fruit,and exploring the regulation mechanism of As A synthesis is the basis of As A improvement in apple.The previous study of the research group found that the expression of AMR1（ascorbic acid mannose pathway regulator 1）homologous gene in apple was negatively correlated with the content of As A.This paper further analyzed the relationship between the expression of AMR1 homologous gene in apple and As A,obtained the transgenic apple lines of Md AMRL1.1 and Md AMRL1.2,and analyzed the change of As A content and its mechanism in transgenic apple lines.The main results were as follows:1.Transcriptome and proteomics studies in’Greensleeves‘apple have found that there are seven AMR1 homologous genes expressed in apple.Compared with tissues with high As A content like leaves,highly homologous Md AMRL1.1 and Md AMRL1.2 were highly expressed in fruit with low As A content.In the process of fruit development,the m RNA and protein expression level of Md AMRL1.1 were negatively correlated with As A content,and could be inhibited by light.This implies that the low As A content of apple may be associated with the high expression of Md AMRL1.1.Full-length sequences of Md AMRL1.1 and Md AMRL1.2 genes,which were significantly related to the accumulation of ascorbic acid in fruit,were cloned from’gala‘apple.Md AMRL1.1 included a 1275 bp ORF（open reading frame）encoding 284 amino acids.Md AMRL1.2 contains an ORF of 1254 bp,encoding 277amino acids,both of which contain F-box motif.In addition,no transmembrane domain was found in Md AMRL1.1/1.2.Studies on its subcellular localization showed that AMRL1.1/1.2was located in the cytoplasm and nucleus.2.We designed Md AMRL1-specific fragments for VIGS（Virus-induced gene silencing）experiments in’Starking Delicious‘fruit in young fruit（75 DAB）.The results showed that,compared with the control group p TRV2,the transcription and protein level of Md AMRL1were significantly decreased in p TRV2-Md AMRL1-treated samples at 6 days,and the As A level was increased.The As A content of Ag NO₃ staining also confirmed that the content of As A in the injection area was significantly increased.3.In order to further verify the function of Md AMRL1.1/1.2 in regulating As A content,we conducted stable genetic transformation test in apple and transgenic apple plants including overexpressed lines and RNAi lines were obtained.Studies on overexpressed plants showed that,after overexpression of Md AMRL1.1,m RNA and protein levels of Md AMRL1.1 increased,ascorbic acid content in leaves decreased significantly,but the content of sugar and the intensity of photosynthesis remain unchanged.However,overexpression of Md AMRL1.2 had no significant effect on As A content.At the same time,RNAi lines were also obtained.It was found that the m RNA and protein levels of Md AMRL1.1 in the leaves of the co-suppressing lines decreased,ascorbic acid content increased slightly.QRT-PCR analysis of the expression of key genes in the As A synthetic and regulatory pathway in transgenic lines showed that overexpression of Md AMRL1.1 did not significantly inhibit the expression of As A synthetic gene,but increased the expression of some key synthase genes such as GMP1.4.To figure out the molecular mechanism of the impact of Md AMRL1.1 on the As A content in apple leaves,transcriptome analysis was performed on three overexpressed lines.We found that,the overexpression of Md AMRL1.1 caused the 154 genes raised,85 genes suppressed in transgenic apple,but the expression of most genes related to the As A synthetic metabolism and regulation has no obvious change,and the key genes in As A biosynthetic pathway such as GMP1,GME1,MIXO,and ERF98,a transcription factor related to the regulation of As A synthesis,were significantly up-regulated.In virus-induced Md AMRL1silencing test in apple fruits,the expression of GMP1 protein increased significantly.Western Blot analysis of GMP1 protein in leaves of overexpressed lines showed that GMP1protein expression was down-regulated.Bimolecule fluorescence hybridization results showed that AMRL1 could interact with GMP1,and MG132 could enhance the interaction intensity.These results indicate that AMRL1.1 could interact with GMP1 and regulate the level of GMP1 protein through ubiquitination,and then negatively regulate the synthesis of As A in apple.Meanwhile,the upregulation expression of As A synthetic gene such as GMP1in Md AMRL1.1-overexpressed transgenic apple may be a feedback regulation mechanism of As A synthesis.