Molecular Mechanism Of Leaf Color Formation In Lagerstroemia Indica With Purple Leaves

Leaves of most plants are green;while red(purple)-leafed plants exist in almost every species throughout the plant kingdom.Red(purple)-leafed plants not only have higher stress resistance and light protection ability,but also play an important role in landscape architecture.The formation of red(purple)leaves in most plants is due to the accumulation of anthocyanins.Crape myrtle(Lagerstroemia indica),a famous summer flowering woody plants in the world,has high ornamental value and is widely used in gardens.Flowers and leaves of purple-leafed crape myrtle can be appreciated together,which enriches the ornamental value of crape myrtle.However,the molecular mechanism of purple leaf formation in crape myrtle is not clear.In order to demonstrate the molecular mechanism of purple leaf formation of crape myrtle,L.indica‘Ebony Embers’(purple leaf)and‘Arapahoe’(green leaf)were selected as materials.The genetic model of purple leaf traits,the metabolite basis of change of leaf color,and the regulation mechanism of anthocyanin synthesis in leaves of crape myrtle were analyzed by the methods of genetics,metabolomics and transcriptomics.The key transcription factors for the regulation of anthocyanin synthesis in crape myrtle leaves were found,and their functions were verified.The main results are shown as follows:1.Two populations,‘Arapahoe’(green leaf)בEbony Embers’(purple leaf)and‘Pocomoke’(green leaf)בEbony Flame’(purple leaf),were obtained by crossing purple-leafed and green-leafed crape myrtle cultivars.Taking two hybrid populaiotns as materials,the L~*,a~*and b~*parameters and anthocyanin content of leaves were compared,and the accumulation of anthocyanin was determined to be the key for purple leaf formation of crape myrtle.The major gene plus polygene inheritance model was used to investigate the inheritance of anthocyanin content in hybrid generations.The results showed that the inheritance of anthocyanin content in leaves of crape myrtle was controlled by two pairs of major genes,and was less affected by environment.2.The comparison of flavonoid metabolites in leaves of two cultivars showed that the main reason for the high accumulation of anthocyanins in purple leaves is the high-level accumulation of most metabolites in flavonoid pathway,while the contents of flavonoid metabolites in green leaves decreases significantly with the development of leaves.The main pigments in leaves of crape myrtle were cyanidin derivatives and pelargonidin derivatives,which are cyanidin O-syringic acid,cyanidin 3-O-galactoside,cyanidin 3,5-O-diglucoside,peonidin 3-O-glucoside chloride and pelargonidin 3-O-Glucoside.3.The analysis of transcriptome data showed that most anthocyanin synthesis structural genes were continuously highly expressed in purple leaves,while the expression significantly decreased at S3-S5stage in green leaves.Correlation analysis between five differentially accumulated anthocyanins and differentially expressed genes showed that the expression levels of several anthocyanin structure genes,hormone response genes,circadian rhythm response genes,MYB and b HLH transcription factors were highly correlated with contents of cyanidin O-syringic acid,cyanidin 3-O-galactoside,cyanidin 3,5-O-diglucoside,peonidin 3-O-glucoside chloride.Two hub modules were identified in the weighted gene coexpression network analysis(WGCNA)of differentially expressed genes.In the two modules,HY5,HYH,MYB,b HLH and CDC5 and ATHB-8 transcription factors are the key genes.HY5,HYH,MYB and b HLH were selected by correlation analysis and WGCNA analysis,which is inferred that they may be the key genes for the regulation of anthocyanin synthesis in crape myrtle leaves.4.Comparing the anthocyanin contents and gene expression in leaf color separation population,it was showed that the anthocyanin contents in purple-leaf progenies were about 9 times higher than those of green-leaf progenies,and the expression of Lfi HY5 and Lfi MYB75 in purpl-leaf progenies were about5 times higher than those of green-leaf progenies.Correlation analysis results showed that the expression levels of Lfi HY5 and Lfi MYB75 were significantly positively correlated with the accumulation of anthocyanin in crape myrtle leaves.Transient overexpression of Lfi MYB75 in tobacco promoted the accumulation of anthocyanin,and transient silencing of Lfi HY5 and Lfi MYB75 in purple-leaf crape myrtle reduced the content of anthocyanin by 40%.The results of yeast two-hybrid(Y2H),yeast monohybrid(Y1H)and dual-luciferase reporter assays confirmed that Lfi HY5 can activate the expression of Lfi MYB75 to active transcription of anthocyanin structural genes Lfi CHS and Lfi ANS,so as to enhance the accumulation of anthocyanins.Lfi MYB75 and Lfib HLH1/4 could form a complex to enhance the activation of expression of Lfi CHS and Lfi ANS.Through cloning and sequencing,it was found that there were three haplotypes of Lfi HY5:Lfi HY5a,Lfi HY5g and Lfi HY5p in crape myrtle.The results of Y1H and dual-luciferase reporter assays confirmed that only Lfi HY5a and Lfi HY5p activated Lfi MYB75,and Lfi HY5g did not activate Lfi MYB75.Based on the results of our investigation,the increase of anthocyanin is the key reason for the leaf color formation in purple leaves of crape myrtle.The anthocyanin content was controlled by two pairs of major genes,and the anthocyanin hyperaccumulation in purple leaves is due to the continuous high expression of structural genes in the whole flavonoid biosynthesis pathway.It is found that Lfi HY5 and Lfi MYB75 are key regulatory genes for anthocyanin synthesis in crape myrtle leaves.Lfi HY5 can positively regulate the transcription of Lfi MYB75 to activate the transcription of anthocyanin structural genes Lfi CHS and Lfi ANS to promote the accumulation of anthocyanins,and Lfib HLH1 or Lfib HLH4 can form a protein complex with Lfi MYB75 to enhance the activation of Lfi CHS and Lfi ANS.Lfi HY5 is located in the upstream position of the above regulation model,which determines the expression level of Lfi MYB75.This indicates that Lfi HY5 is in a more critical position in the anthocyanin synthesis of crape myrtle leaves,and its expression level plays a very important role in leaf coloration of crape myrtle.The results not only provide a theoretical basis for further understanding the anthocyanin synthesis and regulation mechanism,but also provides technical support for the directional cultivation of new varieties with various leaf color of crape myrtle.