Research on application exogenous abscisic acid in inhibiting early flowering and associated genes expression characteristics in blueberry

Abstract: 【Objective】The study aimed at investigating the effect of abscisic acid on flower bud dor-mancy and related genes expression in blueberry (Vaccinium spp.).【Methods】The chilling accumula-tion of blueberry in winter was calculated using the Utah model in Jinhua. Annual branches of 20 cm length with flower buds of Southern highbush blueberry cultivar‘Emerald’were collected at the blue-berry base for the study of sprouting rate estimation, hormone treatment, and cold treatment following hormone treatment. The branches with flower buds were randomly divided into three replicates. Sprout-ing rate was used as an indicator to determine dormant states of the flower buds and chilling require-ment for breaking dormancy.‘Emerald’blueberry branches with flower buds were sampled on Novem-ber 20, 2019, December 11, 2019 and January 8, 2020. Flower buds in different dormancy states were used as experimental materials which were treated with exogenous ABA (50, 100, 150 μmol · L^{- 1}) and different chilling times following ABA treatment. We aimed at investigating the effects of ABA and chilling time on dormancy promotion or release on blueberry floral buds. The qRT-PCR was employed to explore the expression of dormancy related genes in blueberry flower buds with treatments of exoge-nous ABA and different chilling times. The total RNA of‘Emerald’flower buds was extracted by modi-fied CTAB method. Reverse transcription of the total RNA into cDNA was performed by HiScript Ⅲ RT reagent Kit. Eight genes, VcNCED3, VcPYL1, VcPYL2, VcSVP, VcCBF, VcICE, VcGA20OX-1 and VcFT were filtered from differently expressed genes of a transcriptome profile of flower bud dormancy-release. The specific primers of those genes in blueberry were designed by Primer 5.0 software. The two- steps qRT- PCR was performed with four biological replications per sample using a 2 × Sybr Green qPCR Mix (High ROX) kit in the following system including2×SybrGreenqPCRMix of 10 μL,5 μmol forward primer and reverse primer respectively, cDNA (200 ng·μL-1) 1 μL, ddH2O 8 μL, com-posing a 20 μL total volume. The relative expressions of genes were estimated using 2-ΔΔCt method. The significant differences were detected using SPSS Statistics 21 software. The data results were analyzed using single factor analysis of variance, such as LSD, Duncan, etc., and using prism software for plot-ting.【Results】The endo-dormancy of flower buds was broken on between December 4 and December 11 in 2019. The sprouting rate of flower buds on December 11 in 2019 reached 83.14%, which indicat- ed the endo-dormancy was broken based on an arbitrary release standard of 50%. Chilling requirement of‘Emerald’was 177.33 C·U. After that point, flower buds of blueberry had strong sprouting potential and high germination rate before entering eco-dormancy phase. Accordingly, we defined an endo-dor-mancy state, endo-dormancy release state and eco-dormancy state for blueberry variety‘Emerald’flower buds on Nov. 20, Dec. 2019 and Jan. 8, 2020. Exogenous application of ABA treatment before eco-dormancy state could significantly inhibit sprouting of flower bud and promote flower bud into endo-dormancy. The germination rates of flower buds gradually decreased with the increase of ABA concen-tration. The exogenous application of 150 μmol·L-1 ABA during the endodormancy state could signifi-cantly inhibit the germination of flower buds. The results showed that with the increase of 4 ℃ cold treatment time, the germination rate of flower buds increased significantly, the accumulation of 8 days low temperature could counteract the inhibition of ABA on germination. At the same time, cold treat-ment could also promote the uniformity of flowering. According to the Utah model, chilling accumula- tion at 4 ℃ for 4, 8 and 12 days is equivalent to 96, 192, 288 C · U which could achieved in the field. However, no inhibitory effect was found when exogenous application of ABA was done in ecodorman-cy state. The expression level of the VcNCED3 in dormancy flower buds was significantly up-regulated after ABA treatment, and the expression level of the VcNCED3 was promoted by 3.67 fold. It was sig-nificantly down-regulated with low temperature breaking dormancy, the expression reached 0.15 when treated at 4 ℃ for 8 days. The expression profile of the VcPYL1 showed similar pattern with the VcNCED3. By contrast, the genes VcPYL2, VcCBF, VcGA20OX-1 and VcFT were down-regulated with exogenous ABA treatment probably due to promoting flower buds into dormancy and being up-regulat-ed by low temperature to break dormancy. Our results suggested that the genes might be involved in the regulation of blueberry flower bud dormancy.【Conclusion】The exogenous application of ABA could promote blueberry flower buds into deep endo- dormancy state through regulating the expression of flower buds dormancy related genes and inhibit blueberry early flowering.