Molecular Mechanism Of F-box Protein MdAMR1L1 Regulation In Apple Ascorbic Acid Content

Ascorbic acid(Asc)is an important antioxidant in plants and humans that plays important roles in various physiological processes.Understanding the regulation of Ascorbic acid level in fruit plants is important for improving fruit quality and plant resistance resiliency.Previously,we have found that both the transcript level and protein abundance of F-box protein Ascorbic Acid Mannose pathway Regulator 1 Like 1,MdAMR1L1 was negatively associated with Asc levels in apple(Malus×domestica)fruit.In this study,MdAMR1L1 transgenic apple seedlings were used as the materials.The molecular mechanism of MdAMR1L1 regulate apple Asc content was carried out from the relationship between MdAMR1L1,Asc biosynthetic enzyme GDP-mannose pyrophosphorylase MdGMP1 and ethylene response factor MdERF98 and their functions in Asc synthesis.The purpose is to clarify the molecular mechanisms involved in the perception and/or transduction of internal signals for the regulation of Asc content,enrich the theoretical basis of Asc synthesis regulation,and provide theoretical guidance for improving the Asc level in apple fruit and improve fruit quality.The main findings are as follows:1.The F-box protein MdAMR1L1 promoted MdGMP1 protein degradation via the ubiquitination pathway to regulate apple Asc synthesis at the post-translational level.Studies on Asc synthesis and regeneration in MdAMR1L1 transgenic apple leaves showed that the expression of MdAMR1L1 gene was negatively correlated with the apple Asc content.Overexpression of MdAMR1L1 reduced Asc content,but promoted the transcription of key genes of L-Gal pathway,especially MdGMP1.The transcription of regeneration genes was not affected.This is consistent with previous results.To further explore the regulatory mechanism of MdAMR1L,it was discovered that MdGMP1 protein may interact with it through IP-MS/MS technology.The MdGMP1 protein abundance and enzyme activity during fruit development were relative to Asc content.Compared with the WT,MdAMR1L1overexpression inhibited the enzyme activity of GMP and the accumulation of its catalytic product GDP-D-mannose(GDP-D-Man)in apple leaves,while silencing MdAMR1L1promoted those.The results showed that the Asc content in MdAMR1L1 transgenic apple lines was related to the abundance of MdGMP1 protein.Yeast two hybrid and immunocoprecipitation confirmed the interaction between MdAMR1L1 protein and MdGMP1 protein.The results of protein ubiquitination and degradation analysis showed that MdAMR1L1 directly targeted MdGMP1 protein and promoted its degradation through ubiquitination pathway.In conclusion,apple F-box protein MdAMR1L1 interacts with MdGMP1 and promotes MdGMP1 protein degradation via the ubiquitination pathway to negatively regulate apple Asc synthesis at the post-translational level.2.Transcription factor MdERF98 participates in apple Asc biosynthesis by positively regulating the transcription of MdGMP1.To explore the mechanism of the significant up-regulation of MdGMP1 m RNA levels in apple leaves overexpressing MdAMR1L1,transcriptional analysis found that the transcriptional levels of MdGMP1 candidate regulatory factor MdERF98 were also significantly up-regulated.According to the prediction of cis-acting elements,the promoter of MdGMP1 contained one DRE core sequence(ACCGAC)for ERF transcription factor binding.Experiments verified that MdERF98directly binds to the MdGMP1 promoter and positively regulated the transcription of MdGMP1.Overexpression of MdERF98 increased the transcription of MdGMP1 and GDP-mannose-3',5'-epimerase(MdGME1)and Asc level in transgenic callis,while silencing of MdERF98 gene decreased the transcription transcription of MdGMP1 and MdGME1 and Asc level in transgenic apple leaves.We suppressed the expression of MdERF98 in WT and MdAMR1L1-OE transgenic apple leaves.The results showed that the silencing of MdERF98gene resulted in the down-regulation of MdGMP1 and MdGME1 gene transcription,and the Asc level in MdAMR1L1 overexpression transgenic apple leaves was further down-regulated.In conclution,MdERF98 positively regulates the expression of MdGMP1 and MdGME1,and MdGMP1 upregulation in the MdAMR1L1-OE lines was due to increased MdERF98expression,which mitigated the decrease in Asc level caused by the overexpression of MdAMR1L1.3.MdERF98 participates in the feedback regulation of Asc synthesis.To confirm the reason of increasing MdERF98 transcript level in transgenic apple leaves overexpressing MdAMR1L1,we found that the transcriptional level and promoter activity of MdERF98could be inhibited by exogenous Asc.And MdAMR1L1 overexpression transgenic apple leaves treated with exogenous Asc can restore the expression of MdERF98 and MdGMP1.For this,we proposed that MdERF98 might be involved in the feedback regulation of Asc synthesis.In order to verify this hypothesis,we obtained transgenic apple callis in which MdGal DH(L-galactose dehydrogenase),a key enzyme for Asc synthesis,was overexpressed and silenced.It was found that overexpression of MdGal DH increased the Asc level in callis,but inhibited the transcription of MdERF98 and the promoter activity.In MdGal DH silenced callis,the Asc level decreased,the transcription level of MdERF98 increased.The transcriptional level of MdGMP1 in transgenic callis was similar to that of MdERF98.These results further confirm that the expression of MdERF98 is regulated by the feedback of Asc level.MdERF98 participates in the feedback regulation of Asc synthesis,and plays a role in maintaining the steady state of Asc content in apple cells.Overexpression of MdAMR1L1increased the content of H₂O₂ in apple leaves than that in WT.Exogenous Asc treatment decreased the content of H₂O₂,and the change of MdERF98 at the transcriptional level was similar.At the same time,H₂O₂ inducer methyl viologen treatment reduced the Asc level in apple leaves,but promoted the transcription of MdERF89 and MdGMP1.These results showed that the increased MdERF98 and MdGMP1 transcript levels in transgenic apple overexpressing MdAMR1L1 was related to H₂O₂ accumulation.4.bHLH type transcription factor MdMYC2L-6900 negatively regulates the transcription of MdGMP1 and participates in light-regulated apple Asc synthesis.Overexpression of MdAMR1L1 resulted in the up-regulation of the expression of multiple b HLH type transcription factors in apple leaves,including MdMYC2L-6900,which is a homologous gene of MYC2(the key regulator in the jasmonic acid signaling pathway).According to the prediction,the promoters of MdERF98 and MdGMP1 contained the G-box core sequence(CACGTN)for MYC2 binding.The results of interaction experiments showed that MdMYC2L-6900 did not bind to the promoter of MdERF98 but directly binded to the promoter of MdGMP1 and inhibited its transcription.Simultaneously,we found that the expression of MdMYC2L-6900 was inhibited by light and negatively correlated with Asc content.Overexpression of MdMYC2L-6900 increased the transcription of MdGMP1 and Asc content in transgenic apple leaves,while silencing of MdMYC2L-6900 had no obviously effect on it.The results of MV treatment of MdMYC2L-6900 transgenic apple leaves showed that ROS accumulation inhibited the expression of MdMYC2L-6900 and promoted the expression of MdGMP1.MV treatment further reduced the Asc content of MdMYC2L-6900overexpressing apple line.But it had no significant effect on the Asc content and MdGMP1expression in MdMYC2L-6900 silent apple leaves.The results suggest that transcription factor MdMYC2L-6900 participate in the light regulation of Asc synthesis by inhibiting the transcription of MdGMP1,but there may be a functional redundancy of MYC gene family members in apple.