Functional analysis of sucrose synthase PavSS1 regulating sweet cherry fruit ripening

Abstract: 【Objective】Sweet cherry (Prunus avium L.) is an economically valuable horticultural crop that is widely cultivated in temperate regions. Its fleshy fruits have nutraceutical properties and antioxi-dant activity. Sucrose synthase (SS), which catalyzes the reversible conversion of sucrose to monosac-charose, is a key enzyme in sucrose metabolism. The functional characteristics and regulation of PavSS genes during fruit ripening, however, has not been studied widely. In order to provide a theoretical basis for determining the function of sucrose synthase genes (SS) in sweet cherry during sweet cherry fruit ripening, we characterized the biological function of PavSS1, PavSS2 PavSS3, PavSS4, PavSS5, PavSS6 and PavSS7 genes in sweet cherry.【Methods】To isolate PavSS1, PavSS2 PavSS3, PavSS4,PavSS5, PavSS6 and PavSS7 orthologs in sweet cherry, the protein sequences of AtSS1 (AT1G05560),AtSS2 (AT1G73370), AtSS3 (AT3G43190), AtSS4 (AT4G02280), AtSS5 (AT5G20830), AtSS6 (AT5G37180) and AtSS7 (AT5G49190）were used as the query in a BLAST algorithm- based search against the sweet cherry genome (http://cherry.kazusa.or.jp/map.html). The fruit ripening, quantitative real-time PCR were used separately to analyze the expression patterns of PavSS gene families in fruit tissue obtained in different growth and developmental stages. To verify the effect of sucrose on PavSS gene families expression, qRT-PCR was implemented to assess PavSS gene families expression levels with sucrose treatment. To determine the functional characterization of sweet cherry PavSS1 and PavSS6 during fruit ripening, the tobacco rattle virus-induced gene silencing (TRV-VIGS) technique  was used to knock down expression of the PavSS1 and PavSS6 genes in the sweet cherry landrace ‘Brooks’. RT-PCR was performed using cDNA samples from infiltrated fruit at 14 dpi to verify wheth-er the PavSS1 and PavSS6 gene in‘Brooks’was effectively silenced. To further understand the role of PavSS1 and PavSS6 in the regulation of fruit ripening, we analyzed the anthocyanin contents, ABA con- tents, fruit hardness, soluble sugar contents in the TRV::PavSS1- and PavSS6 infected and control fruits. The transcripts of the ripening-related genes, including PavNCED1, PavPG1, PavXYL1, PavPL1, Pav-PAL, PavCHS, PavANS and PavDFR were assessed.【Results】Seven putative full- length mRNA [PavSS1(Pav_sc0000129.1_g1710),PavSS2(Pav_co4071539.1_g010),PavSS3(Pav_sc0000174.1_g950), PavSS4 (Pav_sc0000130.1_g850), PavSS5 (Pav_sc0000174.1_g960), PavSS6 (Pav_sc0000103.1_g1530), and PavSS7 (Pav_sc0001124.1_g380)] were obtained for PavSS with a start codon ATG and a stop co-don TAA in sweet cherry. The expression level of the PavSS1 was high during the fruit development and ripening. The PavSS1 expression increased gradually from 7 days after flowering, reached a peak on 21st day after flowering, and then decreased gradually during the later stage of fruit development and ripening. The PavSS2 expression was low during fruit growth and development, and the expression pattern of the PavSS2 gene was similar to that of the PavSS1 gene. Both the PavSS3 and the PavSS5 genes expressed during the development and ripening of sweet cherry fruit, but the expression levels were relatively low, and the differences in expression levels were not significant throughout the fruit de-velopment and ripening. The expression of the PavSS4 was high in the early stage of fruit development, reached a peak on the 14th day after flowering, then the PavSS4 gene expression decreased, and main- tained in low expression level. The expression of the PavSS6 gene was low in the early stage of fruit de- velopment and increased in the later stage of fruit development, with the highest expression level 49 days after flowering. The PavSS7 gene expressed during fruit development, but the expression level was relatively low. In addition, the expression of the PavSS7 gene showed two peaks on 7th day and 28th day after flowering, and the expression level was relatively low during fruit ripening. Compared with control, the expression of the PavSS1 and the PavSS6 gene were up-regulated at 6 h, and then sig- nificantly down-regulated at 12, 24, 48 h in sweet cherry fruit after sucrose treatment. However, the PavSS2, PavSS3, PavSS4, PavSS5 and PavSS7 genes in sweet cherry fruit did not differ in expression levels from those of the control after sucrose treatment. The expression of PavSS1 and PavSS6 was mark- edly reduced in the TRV::PavSS1 and the PavSS6-infected fruits compared with those of the TRV::00-in- fected fruits, the PavSS1-silenced fruits displayed light red or yellow-white or green-yellow at 21 dpi, the control fruits (TRV::00) fruits had a red and dark red epidermis. However, the fruit phenotypes for the PavSS6-silenced fruits showed no visible defects compared with the control fruits at 21 dpi. Several ripening-related variables in the PavSS1-and PavSS6-silenced and control fruits were analyzed. The re-sults showed ABA content, anthocyanin content, and sucrose content of the TRV::PavSS1-infected fruit were significantly reduced compared with that of the TRV::00- infected fruit at 30 dpi. And in PaMADS7- silenced fruits, fruit firmness and fructose were greater than those of the control fruits. Moreover, the qRT-PCR results showed that the expression of the ripening-related genes, PavNCED1, PavPG1, PavXYL1, PavPL1, PavPAL, PavCHS, PavANS and PavDFR were strongly suppressed in PavSS1- silenced fruits compared with the control fruits. However, compared with the controls, the PavSS6-silenced sweet cherry fruits had no significant differences in ABA content, anthocyanin content, soluble sugar (sucrose, glucose, and fructose) contents, fruit firmness, and transcript levels for the ripen- ing-related genes.【Conclusion】The PavSS1 is most likely to play a pivotal role in the regulation of strawberry fruit ripening, and its expression could be inhibited by sucrose.