Screening Of Key Genes For Apple Russeting Formation And Function Identification Of MdLIM11 Gene

Apple russeting is a non-invasive physiological disease,which occurs in most apple production areas and varieties in China,and seriously affects the yield and quality of apple.At present,bagging is one of the effective measures to prevent apple fruit russeting,but it's not benefical to the sustainability of the apple.To study the key genes involved in the formation of apple russeting and study their functions for the further creation of new russeting free apple germplasm is important.Previous studies proved that the occurrence of fruit russeting is related to the accumulation of lignin and suberin,and a few transcription factors regulating suberin biosynthesis have been screened.However,there is no report on transcription factors regulating lignin biosynthesis in fruit russeting.In this study,bagging and non-bagging ‘Golden Delicious' apples at 30 DAF,60 DAF and 150 DAF(days after flowering)were used as test materials.Though the histological,transcriptomics,proteomics,transcriptome-proteome correlation analysis,as well as gene family analysis and functional verification of the identified transcription factors regulating lignin biosynthesis,the main results are as following:1.For histological analysis,the results showed that there was no fruit russeting in bagging apples,but the skin of non-bagging apples began to form microcracks at 30 DAF,and the epidermal cells began to break;at 60 DAF,the microcracks were further deepened,forming a “V” type crack,and the fruit russeting components began to accumulate.At 150 DAF,the “V” type crack became longer and deeper,and the fruit russeting components deposited in large quantities.2.Transcriptome data showed that 273 differentially expressed genes(DEGs)were common in the three periods,of which 77 were up-regulated and 196 were down-regulated.KEGG pathway enrichment results revealed that 114 DEGs were annotated into 72 pathways,among which DEGs were involved in a large number of metabolic pathways is phenylpropanin biosynthesis.Proteome data analysis showed that 1,293 differentially expressed proteins(DEPs)were screened,including 447 up-regulated DEPs and 846 down-regulated DEPs,and totally 20 DEPs were common in the three periods,all of which were down-regulated.All DEPs were pathway-annotated and the results showed that the majority of DEPs were annotated into metabolic pathways,secondary biosynthesis and carbon metabolism.3.A total of 10,964 genes and proteins were correlated by transcriptome-proteome correlation analysis,of which 119 were differentially expressed.The results showed that 52 of the 119 DEPs/DEGs were annotated into 10 KEGG pathways,including metabolic pathway,secondary metabolite biosynthesis,phenylpropanoid biosynthesis pathway,etc.Among them,4 DEGs of phenylpropanoid biosynthesis pathway,such as peroxidase(PRX,MD17G1092400 and MD03G1059200),cinnamyl-alcohol dehydrogenase(CAD9,MD08G1009200),shikimate O-hydroxy cinnamoyl transferase(HCT,MD17G1225100)anmay be directly participate in lignin biosynthesis,leading to the formation of fruit russeting.In order to further identified the regulatory factors of these genes,a functional protein association network was constructed in this study,the results showed that the homologous gene(MD15G1068200)of AtLIM1 transcription factor was involved in the regulation of lignin biosynthesis related genes,and directly acted on the key enzyme CAD9 that catalyzing the final step of lignin monomer production,indicating that LIM transcription factor was involved in lignin metabolism.4.The LIM family genes of apple were identified by GDR database and Plaza database.We totally identified 11 LIM family genes and located them on 7 apple chromosomes by genome-wide analysis.According to their positions on the chromosomes they were named MdLIM1-MdLIM11.These genes were divided into four categories by the phylogenetic tree analysis,and it was found that MdLIM11(MD15G1068200)was clustered with NtLIM1,which has been proved to directly regulate the expression of genes that related to lignin biosynthesis.The results of qRT-PCR suggested that the expression level of MdLIM11 in russeting-free fruits was significantly higher than that in russeting fruits,indicating that the transcription factor of MdLIM11 may play a key role in the formation of russeting in apple fruits.5.The results showed that MdLIM11 was located in the cytoplasm and nucleus,had the ability to bind to the promoter of MdPAL,and could specifically bind to PAL-box,inhibiting the expression of MdPAL.When overexpressed MdLIM11 in Arabidopsis thaliana,the activity of PAL and POD reduced,the expression levels of lignin biosynthesis-related genes,such as PAL,C4 H,HCT,CAD and POD were significantly decreased,and the contents of small molecular metabolites of lignin biosynthesis,such as p-coumaraldehyde,coniferaldehyde,coniferyl alcohol,sinapinaldehyde and sinapyl alcohol were also significantly decreased.Above all,we identified a MdLIM11 transcription factor related to apple russeting formation though the transcriptomics,proteomics and transcriptome-proteome correlation analysis.By combining with PAL-box,MdLIM11 negatively regulates the expression of genes involved in lignin biosynthesis,enzyme activities and contents of small molecule metabolites.This study analyzed the molecular mechanism of apple russeting from the perspective of lignin biosynthesis,and provided theoretical reference for the creation of new russeting free apple germplasm.