Article pubs.acs.org/JAFC
Assessment of Sugar Components and Genes Involved in the Regulation of Sucrose Accumulation in Peach Fruit Sornkanok Vimolmangkang,†,§,∥ Hongyu Zheng,†,∥ Qian Peng,† Quan Jiang,# Huiliang Wang,⊗ Ting Fang,† Liao Liao,†,⊥ Lu Wang,† Huaping He,⊗ and Yuepeng Han*,†,⊥ †
Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden of the Chinese Academy of Sciences, Wuhan 430074, People’s Republic of China § Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand # Institute of Forestry and Pomology, Beijing Academy of Agriculture and Forestry Sciences, A12, Ruiwangfen, Beijing 100093, People’s Republic of China ⊗ Institute of Fruit Tree and Tea, Hubei Academy of Agricultural Sciences, 430209 Wuhan, People’s Republic of China ⊥ Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, People’s Republic of China S Supporting Information *
ABSTRACT: Soluble sugar contents in mature fruits of 45 peach accessions were quantified using gas chromatography analysis. Sucrose is the predominant sugar in mature fruit, followed by glucose and fructose, which have similar concentrations. Overall, sucrose metabolism and accumulation are crucial determinants of sugar content in peach fruit, and there is a wide range of sucrose concentrations among peach genotypes. To understand the mechanisms regulating sucrose accumulation in peach fruit, expression profiles of genes involved in sucrose metabolism and transport were compared among four genotypes. Two sucrosecleaving enzyme genes (SUS4 and NINV8), one gene involved in sucrose resynthesis (SPS3), and three sugar transporter genes (SUT2, SUT4, and TMT2) were prevalently expressed in peach fruit, and their expression levels are significantly correlated with sucrose accumulation. In contrast, the VAINV genes responsible for sucrose cleavage in the vacuole were weakly expressed in mature fruit, suggesting that the sucrose-cleaving reaction is not active in the vacuole of sink cells of mature peach fruit. This study suggests that sucrose accumulation in peach fruit involves the coordinated interaction of genes related to sucrose cleavage, resynthesis, and transport, which could be helpful for future peach breeding. KEYWORDS: Prunus persica, soluble sugar, sweetness, sucrose metabolic genes, sugar transporter genes
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INTRODUCTION Sweetness is an important component of fruit organoleptic quality, and its intensity depends on sugar content and composition because different types of soluble sugars have different relative degrees of sweetness.1 Sugar partitioning in fruit is a complex process that initiates the export of photoassimilates via sieve elements of phloem into sink tissues.2,3 During phloem unloading, imported sugars enter surrounding cells through symplastic and/or apoplastic pathways. Sucrose enters the cell as sucrose or as hexoses after hydrolysis by cell wall invertase (CWINV). In sink cells, the transported sugars are either stored or metabolized. Sugar metabolism in cytoplasm comprises a complex regulatory network involving a variety of enzymes, including neutral invertase (NINV), hexokinase (HK), fructokinase (FK), sucrose phosphate synthase (SPS), and sucrose synthase (SUS).4 This metabolic process generates various metabolites, such as glucose-6-phosphate and soluble sugars (glucose, fructose, and sucrose). The former is a precursor for starch synthesis, whereas the latter are mainly transported into the vacuole by special transporter proteins located on the vacuole membrane, such as sucrose transporter (SUT), tonoplastic monosaccharide transporter (TMT), and sugar transporter protein (STP). When sucrose is transported into the vacuole, it © 2016 American Chemical Society
can also be converted to fructose and glucose by vacuolar acid invertase (VAINV). Peach (Prunus persica (L.) Batsch) belongs to the family Rosaceae and is one of the most economically import fruit crops worldwide, especially in the temperate zones. However, peach consumption is tending to decrease due to poor flavor falling short of consumers’ expectation.5 Because sugar accumulation affects both fruit yield and quality, genetic improvement of fruit sweetness is considered an important goal of peach breeding programs.6,7 In peach, sorbitol and sucrose are the main transport forms of photoassimilates, and apoplastic phloem loading forms the major route for exporting carbohydrates from mature leaves.8,9 However, the mechanism of phloem unloading in peach fruit has not been fully elucidated, although a more recent study reveals evidence for an apoplastic sucrose transfer in the early and middle phases of fruit development.10 Peach fruit accumulates various types of soluble sugars, such as sucrose, glucose, fructose, sorbitol, and inositol,11−13 and Received: Revised: Accepted: Published: 6723
May 12, 2016 August 18, 2016 August 18, 2016 August 18, 2016 DOI: 10.1021/acs.jafc.6b02159 J. Agric. Food Chem. 2016, 64, 6723−6729
Article
Journal of Agricultural and Food Chemistry
powder was dissolved in 10 mL of 80% methanol (v/v). The mixture was incubated in a water bath at 75 °C for 15 min, extracted in an ultrasonic bath for 45 min, and then centrifuged at 12000 rpm for 20 min. A total of 980 μL of supernatant was collected, mixed with 20 μL of internal standard solution (2.5% methyl-α-D-glucopyranoside, 2.5% phenyl-β-D-glucopyranoside, and 10% acetone), and then centrifuged at 12000 rpm for 20 min. An aliquot of 0.5 mL of supernatant was transferred to a new 2 mL Eppendorf tube and evaporated at 60 °C for about 2 h. The residue was dissolved in 0.8 mL of hydroxylamine hydrochloride solution in pyridine (20 mg/mL), incubated at 75 °C for 1 h, and then cooled to room temperature. Subsequently, 0.2 mL of trimethylchlorosilane and 0.4 mL of hexamethyldisilazane were added. The mixture was incubated at 75 °C for 2 h and centrifuged at 12000g for 20 min at room temperature. The supernatant was filtered through a 0.22 μm filter. The filtered supernatants were subjected to gas chromatography (GC) analysis using an Agilent 7890A gas chromatograph (Agilent, USA) with a flame ionization detector to estimate sugar contents according to the protocol described by Bartolozzi et al.30 A nonpolar HP-5 MS phenyl-methyl-siloxane column with 0.25 μm film thickness (30.0 m long × 0.32 mm i.d., USA) was used for separation. Front inlet and detector temperatures were 270 and 300 °C, respectively. The flow rates of H2, N2, and air were 25, 30, and 400 mL/min, respectively. The sample volume injected was 1 μL, and the split ratio was 30:1. The temperature program started at 130 °C and was set to rise to 198 °C at 6 °C/min, then at 0.5 °C/min to 205 °C, and then 20 °C/min to the final temperature of 280 °C, at which it was maintained for 4 min. Sugar concentrations were determined with standard curves, and all standards were purchased from Sigma (St. Louis, MO, USA). Two technical replicates were performed for each sample. Identification of Genes Involved in Sugar Metabolism and Transport in Peach. We initially conducted a search of the peach genome annotation database to identity sugar metabolism genes,31 including CWINV, SUS, NINV, SPS, and VAINV, and sugar transporter genes, including SUT, STP, and TMT. The DNA coding sequence of these genes was further compared against the peach draft genome using BlastN, with an E value cutoff of 1e−5, and no additional member was identified for any gene family. To validate the annotation of these sugar metabolism and transporter genes, they were BLASTed against GenBank’s nonredundant protein (NR) and SwissProt protein databases using BLASTx, with an E value cutoff of 1e−5. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR). Total RNA was isolated using the Universal Plant Total RNA Extraction Kit (BioTeke, Beijing, China), following the manufacturer’s instructions, and DNase I (Takara, Dalian, China) was used to remove any contamination of genomic DNA from RNA extracts. The concentrations of total RNA were measured using a NanoDrop 2000 UV−vis spectrophotometer (Thermo Scientific, USA) and then adjusted to approximately 50 ng/μL by dilution with double-deionized water. The synthesis of cDNA templates was performed using cDNA Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer’s instructions. A SYBR Green-based qRT-PCR assay was carried out in a 10 μL reaction mixture containing 50 ng of template cDNA, 0.1 μM of each primer, and 5 μL of 2 × SYBR Green I Master Mix (Takara, Dalian, China). Amplifications were performed using Applied Biosystems stepone plus Real-Time PCR Systems (Applied Biosystems, USA). The amplification program consisted of one cycle of 30 s at 95 °C, followed by 40 cycles of 10s at 95 °C, and a final step of 40 s at 60 °C. Melting curve analysis was performed at the end of reaction. A peach gene GADPH was used as an internal control.32 Transcripts were quantified using the standard curve method. Differences in cycle threshold (Ct) between target and GADPH genes were used to estimate the relative transcript level of the target gene and calculated as 2 exp−(Cttarget− CtGADPH). All analyses were repeated three times using three biological replicates. Primer sequences used for real-time PCR are listed in Table S2. Statistical Analysis. Mean values were calculated by three replications using Microsoft Excel. Sigmaplot 10.0 software was used to analyze the variation of individual sugar components among
shows variation in sugar composition throughout fruit development.14−17 In immature fruit, glucose and fructose constitute the major sugars, whereas sucrose represents the predominant soluble sugar in ripe fruit. Sorbitol content changes less and remains relatively low throughout fruit growth. To elucidate the mechanism of sugar accumulation in peach fruit, many studies have been conducted on enzymes involved in sugar metabolism. Some research suggests the enzymatic control of sugar content in fruit,18−21 whereas a recent study proposes a poor correlation between the enzymatic capacities and sugar contents.22 In addition, genetic mapping was also extensively carried out to identify key genes responsible for total and individual sugar contents in peach fruit.23−29 Several quantitative trait loci (QTLs) with minor effects were detected, but no candidate genes have been isolated. Overall, our knowledge of the mechanisms underlying sugar accumulation is too limited to be used for the breeding of fruit varieties that meet consumers’ expectations. Peach is indigenous to China, where >10 million tonnes of peach and nectarine is produced annually, thus accounting for approximately 50% of total world production. In this study, we report on the assessment of sugar composition in mature fruits of 45 peach accessions. The relationship was also investigated between sugar contents in fruit and expression levels of seven gene families involved in sugar metabolism or related to vacuolar sugar transport. Our study is not only helpful for future peach breeding but also provides knowledge that is useful for understanding the mechanism of sugar accumulation in fruit.
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MATERIALS AND METHODS
Plant Material. Forty-five peach accessions used in this study are grown at Wuhan Botanical Garden of the Chinese Academy of Sciences, Wuhan, China. Of the 45 accessions, 34 are from China, 10 from Japan, and one from the United States (Table S1). Depending on the harvest date, all tested accessions were divided into categories of early, mid, and late harvesting dates. Fruit characteristics also varied among the selected peach accessions, including flesh color and flesh texture. All of these accessions were grafted on Maotao rootstock (P. persica) and planted at a spacing of 1.5 m × 2.5 m in 2008. Trees of different accessions were trained to the open vase system, with two or three main branches. Trees were grown under standard conditions of irrigation, fertilization, and pest and disease control. For each accession, the maturity date was extrapolated from previous records. Fruits were considered to be mature when they no longer increased in size, softened, and were easily detached and the green color (background color) of the fruit skin disappeared. Mature fruits were randomly collected by a single person to maintain consistency of maturity grade in 2014. Each accession had three replicates, and each replicate consisted of five fruits. Fruit samples were peeled, and pulps were cut into small pieces, immediately frozen in liquid nitrogen, and then stored at −75 °C for later use. In addition, two sweet cultivars, Qianqu and Dubaifeng, with high sugar/acid ratios of 12.5, 10.6, respectively, and two sour cultivars, Legrand and Ruiguang 27, with low sugar/acid ratios of 4.8, and 5.3, respectively, were selected for qRT-PCR assay. Fruit samples were harvested at two stages: the pit hardening stage and mature stage. Fruit samples at the pit hardening stage were collected at 38, 25, 25, and 38 days after full bloom (DAFB) for Qianqu, Dubaifeng, Legrand, and Ruiguang 27, respectively. For mature stage, fruit samples of Qianqu, Dubaifeng, Legrand, and Ruiguang 27 were collected at 127, 93, 105, and 127 DAFB, respectively. To facilitate description, fruit at the pit hardening stage was designated immature fruit (hereinafter the same). Measurement of Sugar Contents in Peach Fruits. Fruit samples were ground into fine powder in liquid nitrogen using an A11 Basic Analytical Mill (IKA-Werke, Staufen, Germany), and 1 g of 6724
DOI: 10.1021/acs.jafc.6b02159 J. Agric. Food Chem. 2016, 64, 6723−6729
Article
Journal of Agricultural and Food Chemistry
texture or the same flesh color, suggesting that flesh color and flesh texture are also not determinants of peach fruit sugar content. Expression Profiling of Genes Associated with Sucrose Metabolism and Transport in Peach Fruit. As mentioned above, sucrose metabolism and accumulation are of particular interest with respect to fruit quality. In the cell, sucrose is cleaved to fructose and glucose by CWINV, SUS, and NINV, and sucrose resynthesis occurs simultaneously whereby SPS plays a key role.17,35 Cytosolic sucrose is transported into the vacuole by potential sugar transporters, such as SUT, STP, and TMT.10,36 Upon entrance into the vacuole, sucrose is stored or cleaved to fructose and glucose by VAINV. Each gene family involved in sucrose metabolism and transport consists of multiple isoforms in peach, and little is known about the role of different isoforms in sucrose accumulation.7 In this study, 6, 6, 8, 4, 2, 3, 2, and 4 copies of CWINV, SUS, NINV, SPS, VAINV, SUT, STP, and TMT genes, respectively, were identified according to the peach genome annotation.31 To get insight into the role of these genes in sucrose accumulation, their expression profiles were investigated in both immature and mature fruits of four cultivars, Dubaifeng, Qianqu, Ruiguang 27, and Legrand. For the six sucrose-cleaving enzyme genes, SUS1 and SUS4 were predominantly expressed in mature and immature fruits of all tested cultivars, with the expression of SUS4 being significantly up-regulated in fruit at maturity stage (Figure 2). Previous studies demonstrated that SUS enzyme activity shows constant or a slight increase during peach fruit ripening.22,35 Here, our result shows that the SUS4 expression is positively correlated with sugar accumulation. Therefore, it seems that the SUS4 gene may play an important role in sugar accumulation during fruit ripening. Among the eight NINV genes, six, NINV1, NINV2, NINV3, NINV4, NINV7, and NINV8, were expressed in all tested fruit samples, and their expression levels were higher in mature fruits of most tested cultivars than in immature fruits. However, the transcript levels of NINV5 and NINV6 were extremely low or at undetectable levels in immature and mature fruits of all tested cultivars. Of the two VAINV genes, VAINV2 was the predominant transcript in immature fruit, although its expression level decreased to very low or undetectable level in mature fruit. Of the eight CWINV genes, CWINV1, CWINV2, and CWINV3 were expressed in immature and mature fruits of all tested cultivars, whereas the transcript levels of CWINV3 and CWINV6 were extremely low or undetectable. CWINV5 was expressed only in immature fruit of cv. Legrand. For SPS genes, transcript accumulation of SPS3 showed the highest level, followed by SPS4, whereas SPS1 and SPS2 were relatively weakly expressed. The transcript level of SPS3 in mature fruit was significantly higher than in immature fruits. For the three sugar transporter genes, SUT4 was the predominant SUT transcript in fruits, and its expression level significantly increased in mature fruit when compared with immature fruit (Figure 3). SUT2 showed a low level of expression in immature fruit, but increased significantly in mature fruit. Of the two sugar transporter protein genes, STP2 was predominantly expressed in immature and mature fruits of all tested cultivars. Among the four TMT genes, TMT2 was predominantly expressed in immature and mature fruits of all tested cultivars, and its expression level was significantly higher in mature fruit than in immature fruit. In contrast, the transcript
different cultivars. The correlation analysis was performed using software SPSS 16.0 for Windows (SPSS, Chicago, IL, USA). Correlations between experimental variables were measured using Pearson’s correlation coefficients, and significance level was estimated using a two-tailed test. If the p value was
