Studies on fruit coloration and expression of anthocyanin biosynthesis genes in mango cultivar ’Tainong No.1' under different ecological conditions

Abstract: 【Objective】Mango is a famous tropical fruit crop. The external color of fruit is one of the mostimportant factors determining consumers' preference. The principal pigments in the pericarp of mangofruit are chlorophylls,carotenoids,flavonoids and anthocyanins,which are synthesized via phenylpro-panoid pathways. The concentration of fruit anthocyanins is determined by both genotype and environmentfactors including temperature,sunlight,UV and so on. To evaluate the impacts of ecological conditionson the coloration mango(Mangifera indica Linn.) fruit,pigment concentrations and the expressions ofgenes involved in anthocyanins synthesis in the pericarp of mango sampled in Panzhihua,Sichuan prov-ince and in Leizhou,Guangdong province were determined. The results will bring an understanding of theimpacts of ecological conditions on fruit coloration,and provide theoretical basis for production of highquality mango.【Methods】In the present study,‘Tainong No.1'mango samples collected from Panzhi-hua,Sichuan province,and Leizhou,Guangdong province in 2012 were used to study the effects of eco-logical conditions on fruit coloration,pigment contents and the expression genes involving anthocyaninsynthesis. Climacteric data including temperature(the highest temperatures,the lowest temperatures,diurnal temperature differences) and rainfall were collected from weather website(http://www.weather.com.cn). Levels of chlorophylls,caroteniods and flavonoids in the pericarp were measured by traditional meth-ods. Tissues were extracted with extraction solution(80% acetone solution for chlorophylls and caro-teniods,1% HCl methanol for flavonoids),and pigment levels were detected with spectrophotometer. Onegram tissue was extracted with 5 mL extraction solution(0.05% HCl in methanol) at room temperature indarkness for 12 h,then the total anthocyanins were determined by the p H differential spectrophotometrymethod,which involves the measurement of the absorbance at 510 nm and 700 nm on samples dilutedwith p H 1.0 and 4.5 buffers. The chromatic values(L*,a*,b*) were measured by a TC-P II G type auto-matic colorimeter,and chrome and hue angle were calculated base on a*and b*values. Total RNA was ex-tracted using CTAB method. First-strand c DNA was synthesized from 1 μg of total RNA(final volume of 15 μL) using the Primer Script?RT reagent Kit with g DNA Eraser(Taka Ra,Japan) according to the manu-facturer's instructions. The c DNA was diluted five to ten folds and 1 μL of the diluted c DNA was used asthe template for real-time quantitative PCR(Q-PCR) analysis. Primer pairs were designed using PrimerPremier 5 based on NCBI and transcriptome sequences of mango fruits. The Q-PCR mixture(20.0 μL total volume) contained 10 μL of 2X Dy Namo Color Flash SYBR master mix(Thermo Scientific),1.6 μLprimer pairs(10 μmol·L^-1),1 μL template,and 7.4 μL RNase-free water. The reactions were performedon a Light Cycler 480 instrument(Roche,Diagnostics) with Dy NAmo? Color Flash SYBR Green q PCR Kit(Thermo Scientific) according to the manufacturer's instructions. The PCR conditions were as follows: ini-tial denaturation at 95 ℃ for 5 min,followed by 45 cycles of 95 ℃ for 5 s,62 ℃ for 15 s,and 72 ℃ for10 s. The relative expression levels of the target genes were calculated as 2-ΔΔCt and normalized to that ofthe actin gene(Mi ACTIN). The reported values were the averages of three biological replicates.【Results】The results showed that Panzhihua had higher altitudes,average daily highest temperature,average low-est temperature and diurnal temperature differences,but lower rainfall than Leizhou. With regard to peri-carp pigments,the contents of chrolorophylls and carotenoids in fruit sampled in Panzhihua were signifi-cantly higher while the content of flavonoids was lower than in fruit taken in Leizhou. The concentration ofanthocyanins in mango sampled in Panzhihua reached 30.5 mg·kg-1,while they were not detected in sam-ples taken in Leizhou. There was no significant difference in lightness and chrome in the pericarp betweensamples taken in Panzhihua and Leizhou,but the hue angle in fruit from Leizhou was higher than in thosefrom Panzhihua. The expressions of anthocyanin synthesis genes showed significant difference in fruit pro-duced in the two regions. The expression levels of genes involved in anthocyanin synthesis,especially MiPAL,Mi C4 H,Mi CHS1,Mi F3H1,Mi DFR and Mi UFGT,were significantly higher in fruit from Panzhi-hua than those from Leizhou,and the expression levels of Mi C4 H,Mi CHS1,and Mi F3H1,Mi DFR and Mi UFGT in fruit from Panzhihua were over 130,228,63,2.7 and 4.3 times higher than those from Lei-zhou,respectively.【Conclusion】The unique ecological factors,especially high altitude and high diurnaltemperature differences,in Panzhihua favored the color development in‘Tainong No.1'mango as thesefactors enhanced the expressions of anthocyanin synthesis genes especially Mi PAL,Mi CHS1,Mi DFR,and Mi UFGT,and therefore promoted anthocyanin biosynthesis.