Molecular Mechanism On Expression Of Red Leaf Color In Camellia Japonica ‘Jinghua Meinü'

Camellia japonica ‘Jinhua Meinü' is a rare cultivated variety mutated from C.japonica ‘Nuccoi's Bella Rossa'.A gorgeous crimson and large formal double with white stripes.The most significant feature is its glossy foliage color variation from rich purple to red during the leaves sprout,while red juvenile leaves of C.japonica ‘Nuccio's Bella Rossa'generally turn green.Therefore,C.japonica ‘Jinhua Meinü' becomes a prized plant with both ornamental and economic values for its unique leaf color change.In this study,red leaves of C.japonica ‘Jinhua Meinü',coupled with green leaves of C.japonica ‘Nuccoi's Bella Rossa' and Camellia azalea,were all employed to systematically probe metabolites relating to anthocyanin synthesis and carefully research into cloning and characterization of an anthocyanidin synthase gene(ANR)so as to figure out the molecular mechanisms for leaf color variation in C.japonica ‘Jinhua Meinü',which might give an insight into the novel cultivars breeding of colored-leaf ornamental camellias.According to its leaf color changing trend and its growth rate,the leaf sprouting development of C.japonica ‘Jinhua Meinü' was divided into 4 stages(I~IV)to collect the first sprouting leaf within 5 times repetitions.As the results of determination of chlorophyll and carotenoid in leaves by UV spectrophotometry demonstrated,(1)The content of chlorophyll a in C.japonica ‘Jinhua Meinü' at the 4 different stages was respectively 0.282 mg·g-1,0.291 mg·g-1,0.313 mg·g-1 and 0.417mg·g-1,which was up 47.9%,meanwhile,the average content of chlorophyll a in the control group increased from 0.245 mg·g-1 to 0.472 mg·g-1with total 67.4% rising rate.Moreover,the content of chlorophyll b in C.japonica ‘Jinhua Meinü' during the process of leaf growth was 0.134 mg·g-1,0.153 mg·g-1,0.171 mg·g-1 and 0.312 mg·g-1,whose increasing rate was 132.8%.And the average content of chlorophyll b in the control group rose from 0.146 mg·g-1 to 0.312 mg·g-1,in which the growth rate added up to 101.4%.Whatever leaf color changing differed within C.japonica ‘Jinhua Meinü' and the contrast plants,two kinds of chlorophyll both fluctuated in the same trend,and there was no significant difference in the content of chlorophyll a/b in those three different groups measured at the same growth stage.(2)The content of carotenoid in C.japonica ‘Jinhua Meinü' showed a fluctuation from 0.203 mg·g-1 to 0.251 mg·g-1,with 23.6% increasing rate.And the average content of carotenoid in the control was 0.273 mg·g-1,0.319 mg·g-1,0.341 mg·g-1 and 0.360 mg·g-1 respectively,whose rise rate was 31.9%.What's different was that the content of carotenoid in C.japonica ‘Jinhua Meinü'at the four stages was all lower than the content in the control group.Consequently,these results illustrated that there was no great difference in the content of chlorophyll in leaves collected from C.japonica ‘Jinhua Meinü',C.japonica ‘Nuccoi's Bella Rossa' and C.azalea,however,however,the content of carotenoid in leaves from C.japonica ‘Jinhua Meinü' was always lower than the average content in C.japonica ‘Nuccoi's Bella Rossa' and C.azalea,which meant that chlorophyll a/b effected the red color leaf formation insignificantly or chlorophyll and carotenoid affected the red color leaf development slightly.The contents of anthocyanins and polyphenols in leaves from C.japonica ‘Jinhua Meinü',C.japonica ‘Nuccoi's Bella Rossa' and C.azalea were determined through high performance liquid chromatography(HPLC)and UV spectrophotometry.The results showed as follow:(1)The flavonoid extract from leaves of C.japonica ‘Jinhua Meinü' had a conspicuous absorption peak at the area of 500~540nm UV,which could infer that there were numerous anthocyanins in the leaves of C.japonica ‘Jinhua Meinü'.(2)The total content of anthocyanins extracted from the leaves of C.japonica ‘Jinhua Meinü' selected at the four distinct growth stages was much higher than that in the leaves of the control group,namely 20.80-fold,5.40-fold,10.29-fold and 26.40-fold.(3)The content of cyanidin 3-glucoside in the leaves of C.japonica ‘Jinhua Meinü' measured at these four different periods of leaf sprouting was,namely 25.78-fold,5.70-fold,17.28-fold and 94.65-fold,also greatly higher than the average content in the leaves of the control.(4)The total content of polyphenols in the leaves of C.japonica ‘Jinhua Meinü' decreased gradually during the sprouting process.In detail,the percentage of catechin dropped 50%,the percentage of gallocatechin fell 30.77%,meanwhile,there were significant reductions in the contents of epicatechin and epi-gallocatechin,80.95% and 83.33% respectively.But those compounds in the control all increased,which indicated that while the synthetic capability of polyphenol was quite limited in the leaves of C.japonica ‘Jinhua Meinü',epicatechin and epi-gallocatechin,known as two substrates of anthocyanin reductase pathway,dramatically reduced so that this metabolic pathway seemed to be inhibited seriously.The anthocyanin components in the leaves of C.japonica ‘Jinhua Meinü' were tested by using time-of-flight mass spectrometry,6 different anthocyanin components were quantified such as Petunidin 3-O-xylopyranosyl-rutinoside,Petunidin 3-O-glucoside,Delphinidin 3,5-Odiglucoside,Delphinidin 3-O-arabinoside,Cyanidin-3-O-glucoside,Cyanidin 3-(6-pcoumaroylglucoside),in which Cyanidin 3-(6-p-coumaroylglucoside)was in the highest amount.In addition,the other 5 anthocyanin components were firstly figured out in camellias' leaves except for Cyanidin-3-O-glucoside.The expression levels of DFR,ANS,UFGT,LAR,ANR,MYB1 and MYB2 at these four growth stages were analyzed through fluorescent quantitative PCR.As the results illustrated,(1)The expression level of DFR in the leaves from C.japonica ‘Jinhua Meinü' differed insignificantly with that in the leaves from the control,which indicated DFR could express normally at the nearly same level no matter whether the leaves were red or green.(2)The expression levels of ANS and ANR in the leaves from C.japonica ‘Jinhua Meinü' were both obviously lower than that in the control,while the expression levels of LAR and UFGT showed a gradual increase and ran over the express levels in the control at Stage III and Stage IV.Therefore,the synthesis direction of metabolites in falvonoids metabolic pathway shifted when it comes to the leaves of C.japonica ‘Jinhua Meinü',and the express level of ANR referred to polyphenols synthesis pathway in the leaves of C.japonica ‘Jinhua Meinü' was much lower than that in the control,however,the express level of UFGT relating to anthocyanin synthesis was higher than the average level in the control.In consideration of the content change in epicatechin,epi-gallocatechin and anthocyanin glucoside,these consequences demonstrated that anthocyanin synthesis pathway mainly produced anthocyanin pigments,which was quite different from the products in the control.(3)There was no significant difference in the expression level of MYB1 and MYB2 among C.japonica ‘Jinhua Meinü',C.japonica ‘Nuccoi's Bella Rossa' and C.azalea,so the large amount accumulation of anthocyanin in the leaves from C.japonica ‘Jinhua Meinü' had no correlation with the expression level of MYB1 and MYB2.ANR gene sequences were cloned from the leaves in C.japonica ‘Jinhua Meinü',C.japonica ‘Nuccoi's Bella Rossa' and C.azalea by exploiting PCR amplification and genome walking technique derived from cDNA and its genome DNA.(1)After lots of positive bacteria sequencing,two different length ANR sequences(984bp and 1013bp)were successfully obtained from cDNA of the leaves from C.japonica ‘Jinhua Meinü' through several repetitive experiments,which were respectively 30 bp bases and 1bp base more than the sequences cloned in the leaves from the control.(2)The same complete ANR sequence was acquired in genome DNA both from C.japonica ‘Jinhua Meinü' and the control,and this ANR sequence had a 1014 bp ANR ORF,which had 99.93% similarity to the sequence reported in Camellia sinensis.(3)Some parts of the promoter sequence were amplified in genome DNA both from C.japonica ‘Jinhua Meinü' and the control.According to bioinformatics comparison,it was found that there was a long transposon sequence inserted between the TATA box and the transcription initiation site in the genome DNA from the leaves of C.japonica ‘Jinhua Meinü'.This probably blocked the transcriptional expression of ANR.(4)Some parts of a point-mutated sequence referred to ANR was amplified in the genome DNA from C.japonica ‘Jinhua Meinü'.According to the intron model,the point mutation was found to occur in mRNA splicing,which resulted into two different types of ANR.These two ANR sequences were precisely the same as the two ‘abnormal' ANR sequences amplified in the leaves from C.japonica ‘Jinhua Meinü'.The ‘normal' ANR in cDNA was not detected in the leaves from C.japonica ‘Jinhua Meinü' in this study,therefore,it was deducted that the ‘normal' ANR could not express or would express ineffectively when the transposon sequence inserted between TATA box and the transcription initiation site so that it could not be detected while the ‘abnormal' ANR was noticed instead.Because of splice site shift,the transcriptional ANR lost 30 bp bases and then 10 amino acids lost in protein synthesis,which might make anthocyanidin reductase defect so that anthocyanidin could not convert effectively into epicatechin and epi-gallocatechin.Furthermore,there would be large numbers of unstable monomeric anthocyanins accumulated in the leaves,and these anthocyanin-monomers would convert to stable anthocyanins through UFGT pathway,finally the green leaves turned crimson.