Effects of bagging on the accumulation of main carotenoids and the related gene expression in ‘Majiayou’ pomelo

Abstract:【Objective】‘Majiayou’pomelo [Citrus maxima (L.) Osbeck] is an excellent variety in Jiangxiprovince. A bagging treatment was carried out in this study to determine the effects of bagging onthe accumulation of main carotenoids (lycopene and β-carotene), and the expressison change of carotenoidsmetabolism relevant genes and to further understand the relationship between light and pulp carotenoidmetabolism .【Methods】The double paper bags with yellow color on the outside layer andblack color on the inside layer were used for bagging experiment on the fruits of‘Majiayou’pomelo,no bagging treatment was used as the control. The bagging time was July 25, 2017. And the baggedfruits were harvested on August 10, August 25, September 10, September 25, October 10, October 25,and November 10, respectively. The color difference index of the pulp in the equatorial part of‘Majiayou’pomelofruit was measured by“CIELab”color system in Chromatic meter. Both the point near themiddle column and the point far from the middle column were selected from each juice vesicles formeasurement. The items measured included the L*, a*, b*, and H and CCI value. The contents of maincarotenoids (lycopene and β-carotene) in‘Majiayou’pomelo pulp were measured as follow: The extractionof the lycopene and β-carotene from pulp was performed by acetone-petroleum ether method. 2 gsample cutting from the‘Majiayou’pomelo pulp was added into 10 mL mixed solution of acetone-petroleumether (1∶1), Configured samples were set in a ultrasonic cleaner at 50 ℃ for 15 min for sonicatetrearmrnt, then the samples were centrifuged at 8 000 r · min for 10 min. The supernatant was filteredand extracted repeatedly until it was colorless. Extracted sample was transferred to a separatelyfunnel, and was allowed to stand for stratification. The upper organic phase was passed through a funnelcontaining anhydrous sodium sulfate, and the lower layer was extracted again with petroleum ether andevaporated to dryness at 35 ℃. Finally, the sample was ready for HPLC analysis after being dissolvedin 3 mL of dichloromethane and filtered through a 0.45 μm filter. HPLC analysis was carried out by ShimadzuLC-20A Prominence High Performance Liquid Chromatograph using Waters Symmetry ShieldRP18 reversed phase column (4.6 mm × 250 mm × 5 μm) and diode array detector with a detectionwavelength of 450 nm and a column temperature of 30 ℃. The mobile phase was acetonitrile-dichloromethane-methanol (20 - 75 - 5) (V: V: V) and flow rate was 1 mL·min^-1. The β-carotenoid and lycopenecontents were finally detected by using relevant standard. The expressison level of carotenoids metabolismrelevant genes were analyzed by Q-PCR as follow: the total RNA of‘Majiayou’pomelo pulp wasextracted by modified CTAB method, and reverse transcription of total RNA into cDNA was executedby PrimeScript™ RT reagent Kit. Then, specific primers of carotenoids metabolism relevant genes weredesigned by Primer 5.0 software according to its gene sequence. Q-PCR was performed with three biologicalreplications per sample on a bio-rad CFX 96-pcr instrument using a SYBR®Premix Ex TaqTM(Tli RNaseH Plus) kit. And its data were analyzed using a 2^{- Δ Δ ct} method.【Results】The results showedthat the a* value, H value and CCI value of the pulp of the‘Majiayou’pomelo were significantly higherthan those of the CK which indicated that the bagging treatment could improve fruit color and comprehensiveproperties. The lycopene content in the pulp of the bagging treated fruits was significantly higherthan that of the CK and the significant increase of the β-carotene content began on August 25 (30 d afterbagging). Further analysis of the expression of carotenoid metabolism- related genes revealed thatmost of the related genes involved in the synthesis of lycopene and β-carotene were up-regulated at themiddle and early stages of bagging. The up-regulation of PSY2, PDS, β-LCY1 and β-LCY2 related to carotenoidsynthesis was significantly up- regulated by 17.23, 1.72; 8.15, 16.36; 4.56, 1.57; 31.78, 1.17times, respectively, and the BCH gene was associated with β-carotene degradation. At the early stage ofbagging, BCH gene was significantly higher than that of the CK, but it was 3.07 times lower than thatthe non-bagging treatment at the middle stage of the bagging treatmen.【Conclusion】The bagging treatmentcould promot the accumulation of the lycopene and the β-carotene due to the up-regulated expressionof the synthetic genes PSY2, PDS, β-LCY1 and β-LCY2, at the same time the downward expressionof the β-carotene degradation-related gene BCH at the mediumstage of the bagging might also partiallyplay a role in the promotion of th eincrease of the lycopene and the β-carotene.